Device and method for in vivo flow cytometry using the detection of photoacoustic waves

ABSTRACT

A photoacoustic flow cytometry (PAFC) device for the in vivo detection of cells circulating in blood or lymphatic vessels is described. Ultrasound transducers attached to the skin of an organism detect the photoacoustic ultrasound waves emitted by target objects in response to their illumination by at least one pulse of laser energy delivered using at least one wavelength. The wavelengths of the laser light pulse may be varied to optimize the absorption of the laser energy by the target object. Target objects detected by the device may be unlabelled biological cells or cell products, contrast agents, or biological cells labeled with one or more contrast agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. non-provisionalapplication Ser. No. 13/661,551, entitled “Device and Method for In VivoFlow Cytometry Using the Detection of Photoacoustic Waves” filed on Oct.26, 2012, which is a divisional application of U.S. non-provisionalapplication Ser. No. 12/334,217, entitled “Device and Method for In VivoFlow Cytometry Using the Detection of Photoacoustic Waves” filed on Dec.12, 2008, which claims priority from U.S. provisional patent applicationSer. No. 61/013,543, entitled “Device and Method for In Vivo FlowCytometry Using the Detection of Photoacoustic Waves” filed on Dec. 13,2007, all of which are hereby incorporated by reference herein in theirentirety.

GOVERNMENTAL RIGHTS IN THE INVENTION

This work was supported in part by the National Institutes of Healthgrant numbers R01EB000873 and R21EB0005123. The U.S. Government may havecertain rights in this invention.

FIELD OF THE INVENTION

This application relates to a device and methods of using the device tonon-invasively detect laser-induced photoacoustic waves emitted bytarget objects such as cells, pathogens, microparticles, andnanoparticles in vivo, which indicate the presence of target objectscirculating in blood or lymphatic vessels. In particular, thisapplication relates to pulsing circulating target objects with pulses ina broad spectral range from a pulsed laser source, inducing the objectsto emit ultrasonic photoacoustic waves that are subsequently detected byan ultrasound transducer.

BACKGROUND OF THE INVENTION

Flow cytometry (FC) is a well-established diagnostic method that hasrevolutionized cell diagnostics. In this technique, the cells inextracted samples are hydrodynamically induced to flow in single filethrough an artificial nozzle in vitro. Within this artificial flow,individual cells are illuminated by laser light, and thelaser-stimulated fluorescence from molecular probes bound to cellmembrane receptors or light scattered by the cells themselves, isdetected using photodetectors. Multi-color FC with advanced fluorescentprobes is widely used in basic and clinical research, making possiblethe rapid analysis of large populations of cells, the detection of rarecancer cells, and the evaluation of cell viability and drug-cellinteractions. Flow cytometry ordinarily requires invasive extraction ofcells from the living organism, fluorescent cell labeling, and cellsorting procedures, which may lead to unpredictable artifacts such ascytotoxicity. Traditional flow cytometry techniques are not suitable forapplications such as the early diagnosis, prevention, and treatment ofmetastasis, inflammations, sepsis, immunodeficiency disorders, strokes,or heart attacks. Like traditional blood tests, traditional flowcytometry analyzes relatively small volume blood samples. In these smallvolume samples, the detection of rare metastatic cells or other antigensis ineffective until the disease has progressed to a stage in which therare antigens are numerous enough to be detected in small blood samples.The long-term monitoring of cells in their native biological environmentis desired in order to process a larger volume of blood, enabling thedetection of antigens at a much earlier stage in the progression of adisease.

Several in vivo flow cytometry techniques take advantage of the singlefile movement of blood cells through the majority of blood vesselsduring normal circulation. Generally, these in vivo flow cytometrytechniques also utilize light emitted from fluorescent molecular probesto acquire information about the circulating cells, requiring that thecells must be labeled with fluorescent molecular probes.

The powerful fluorescent labeling used in most in vitro and in vivo FCis prone to photobleaching, blinking, or cytotoxicity. These technicalshortcomings limit the extension of traditional FC techniques to thelong-term monitoring of blood or lymph flow on humans in vivo. Thefluorescent labeling of cells may seriously compromise cell function andphysiology. Acridine orange and rhodamine 6G, traditional fluorescentdyes used to label leukocytes in FC, are mutagenic and carcinogenic, aswell as possibly phototoxic. Fluorescent imaging of microvessels withconventional fluorescein isothiocyanate-dextran (FITC) dye leads toelevated interstitial pressure and altered plasma viscosity. Fluorescentdyes or tags used in FC may significantly distort the measuredoccurrence and elimination of cells in circulation, such as apoptotic orcancer cells. The numerous shortcomings associated with the use offluorescent dyes and tags emphasize the need for alternative approachesfor the application of in vivo flow cytometry techniques to clinical orexperimental measurements.

Another in vivo flow cytometry technique under development utilizes thedetection of light scattered from unlabelled cells to deduce informationabout cells in circulation. Although this technique overcomes theshortcomings associated with fluorescent labeling, only a limited subsetof circulating cells are sensitive to light scattering, and there isextensive background noise due to scattered light from red blood cells,which make up the majority of cells in circulation. Further, interveningcells and tissue attenuate the scattered light from the circulatingcells, thereby limiting this technique to cells circulating in vesselsnear the skin's surface.

A novel in vivo flow cytometry technique overcomes most of thechallenges and limitations of the preceding in vivo flow cytometrymethods by utilizing laser-induced photothermal (PT) effects to detectthe presence of target cells in circulation. A target cell is firstilluminated with a pulse of laser light in the visible or near-infrared(NIR) spectral ranges, followed by a second pulse of laser light. Thetarget cell absorbs the energy of the initial laser pulse, inducing alocal temperature rise that distorts the refractive properties of thevolume immediately surrounding the target cell. The characteristics ofthe light from the second laser pulse, as detected by a photodetectorarranged opposite to the laser light source, determine the presence ofthe target cells based on the distortion of the refracted and scatteredlight near the target cell. Although the PT flow cytometry technique maybe used to detect unlabelled target cells, this technique requires thetransmission of light through the vessel to the photodetector on theopposite side. Like all of the other techniques described above thatutilize the detection of light to gather information about circulatingcells this technique is limited to cells circulating in relatively thintissues.

Regardless of the flow cytometry technique, light traveling throughbiological tissues is scattered by surrounding cells and tissues. Assuch, the effectiveness of all of the flow cytometry techniquesdescribed above has been limited to the measurement of cells inrelatively superficial blood vessels, since light may travel for only ashort distance through the cells and tissues surrounding these vesselsbefore becoming too scattered to be detected. A need exists for an invivo flow cytometry technique in which the detected propertiesassociated with the circulating cells are not as readily scattered bysurrounding cells and tissues. Such a technique could be used to detectthe presence of target cells in deep vessels as well as superficialvessels.

One technique used for the study of stationary tissues is photoacoustic(PA) detection. In this detection technique, target cells within thetissue absorb a pulse of light from within the visible or NIR spectrumranges from a laser. The rapid temperature change resulting from the NIRlight absorption by the target cells induces a characteristic ultrasoundPA wave, which travels freely through most biological tissues and isreadily detected by an ultrasound transducer. This technique may be usedfor unlabelled tissues or tissues labeled with various PA contrastagents such as nanoparticles and dyes. However, due to the challenge ofcoordinating the timing and characteristics of the laser illumination,limited sensitivity of the detection of the PA waves, time-consumingsignal-acquisition algorithms, and poor spatial resolution, applicationsof PA imaging methods have been limited to the visualization of largegroups of stationary cells, making this technique inappropriate for therequirements of in vivo flow cytometry.

In vivo flow cytometry techniques to date are limited to the detectionof circulating cells in blood vessels only, due to intrinsic limitationsin sensitivity and resolution. The capability to monitor the traffickingof cells in the lymphatic system would be a valuable additional feature.For example, metastatic malignant cancer cells may spread by way of thelymphatic system, or may form peripheral malignancies in sentinel lymphnodes near the initial tumor. The ability to monitor cells circulatingin the lymphatic system would add a much-needed diagnostic technique foruse in the early diagnosis of a variety of diseases, and for thecontinuous monitoring of many diseases during treatment. In addition,the lymphatic system is a common staging area for most immunologicalphenomena. A need exists to monitor cells circulating in the lymphaticsystem.

A need exists for an in vivo flow cytometry technique that may be usedin superficial or deep vessels, with high sensitivity to individualcells and high resolution to discriminate the relatively rare targetcells from among the numerous surrounding cells. Such a technique willmake possible the non-invasive monitoring of cells in blood vessels aswell as lymph vessels. Further, a need exists for an in vivo flowcytometry technique that measures unlabeled cells, as well as cellslabeled with non-toxic dyes or tags.

SUMMARY OF THE INVENTION

The present invention provides a device for detecting the presence ofmoving target objects such as blood cells within a circulatory vessel ofa living organism at a distance of up to 15 cm away from the vessel. Thedevice includes at least one tunable pulsed laser source thatfunctioning at one or more wavelengths between about 10 Å and about 1cm. The laser pulses emitted by the at least one tunable pulsed lasersource may have a predetermined pulse width that ranges between about0.1 ps and about 1000 ns. The laser pulses emitted by the at least onetunable pulsed laser source may repeat at a predetermined rate rangingbetween about 1 Hz and about 500,000 Hz, and energy fluence of eachlaser pulse may be at a level ranging between about 0.1 mJ/cm² and about1000 J/cm².

The device of the present invention also includes at least one opticalelement that operates to direct a laser pulse from the tunable lasersource to pass through the vessel in which the moving target objects areto be detected. The at least one optical element also focuses laserpulse passing through the vessel into a beam with an elliptically shapedcross-section. The elliptically shaped beam cross-section has a maximumdimension ranging between about 1 μm and about 150 μm.

The device of the present invention also includes at least oneultrasound transducer with a sample rate ranging between about 10 kHzand about 100 MHz to detect the PA signals emitted by the targetobjects.

The present invention further provides a method for detecting at leastone type of moving target object within a circulatory vessel of a livingorganism. The method includes pulsing the target object moving throughthe vessel of the organism vessel with at least one pulse of laserenergy and detecting at least one resulting photoacoustic pulse emittedby the target object. The method also includes analyzing at least onecharacteristic of the detected photoacoustic pulse to determine at leastone characteristic of the detected target objects. The characteristicsof the detected photoacoustic pulse and of the detected target objectsare described in detail below.

The moving target objects may be detected in blood or lymphatic vesselsas far as about 15 cm away from the device of the present invention.Potentially, the moving target objects may be detected anywhere withinthe living organism. In an embodiment, the laser energy pulse may bedelivered at one or more wavelengths ranging between about 10 Å andabout 1 cm, a pulse width ranging between about 0.1 ps and about 1000ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz,and a pulse energy fluence ranging between about 0.1 mJ/cm² and about1000 J/cm². The ultrasonic photoacoustic waves emitted by the targetobjects may be detected at a sample rate ranging between about 10 kHzand about 100 MHz.

The present invention further provides a method for the in vivodetection of a circulating, unlabelled metastatic melanoma cell. Themethod includes pulsing an area of an organism with at least one pulseof near-infrared (NIR) laser energy at a wavelength ranging betweenabout 650 nm and about 950 nm and a laser fluence ranging between about20 mJ/cm² and about 100 mJ/cm², and then detecting the resultingphotoacoustic pulse emitted by the melanoma cell. The method alsoincludes analyzing detected photoacoustic pulses to indicate thepresence of the metastatic melanoma cells in circulation.

The present invention further includes a method of selectivelydestroying target objects circulating in a vessel of an organism invivo. The method includes detecting the target objects circulating inthe vessels, triggering a pulse of laser energy delivered at awavelength and energy level sufficient to cause the destruction of thedetected target object, and monitoring the frequency of detection of thetarget objects circulating through the vessel. When the frequency ofdetection of the target objects falls below a threshold level, thedetection and destruction of target objects is terminated.

Additional objectives, advantages and novel features will be set forthin the description which follows or will become apparent to thoseskilled in the art upon examination of the drawings and the detaileddescription which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the photoacoustic in vivo flow cytometrymethod.

FIG. 2 is a diagram illustrating the in vivo flow cytometry device inaccordance with an embodiment of the invention.

FIG. 3A shows the oscilloscope trace recordings of PA signals from bloodflow in a rat ear vessel with diameter of 50 μm.

FIG. 3B shows the oscilloscope trace recordings of PA signals from skinsurrounding a rat ear vessel before dye injection.

FIG. 3C shows the oscilloscope trace recordings of PA signals from bloodflow in a rat ear vessel 5 min after the injection of Lymphazurin.

FIG. 3D shows the oscilloscope trace recordings of PA signals from theskin surrounding a rat ear vessel measured 20 min after dye injection.

FIG. 4 shows the PA signal detected from the monitoring of the bloodflow in a 50-μm rat ear microvessel with diameter after intravenousinjection of Lymphazurin dye in the tail vein.

FIG. 5 shows the PA signal from circulating GNR in 50-μm rat mesenterymicrovessels as a function of post-injection time.

FIG. 6 is a graph of the normalized number of circulating GNR in bloodmicrovessels of the rat mesentery as a function of post-injection timeand a dashed curve showing averaged data (N=3).

FIG. 7 is a graph of the normalized number of circulating S. aureus inblood microvessels of the mouse ear as a function of post-injectiontime, for bacteria labeled using two different contrast substances, ICGdye and CNT.

FIG. 8 is a graph of the normalized number of circulating E. coli inblood microvessels of the mouse ear as a function of post-injectiontime.

FIG. 9 shows the PA spectra of 50-μm diameter veins in the mouse ear(empty circles), conventional absorption spectra of the B16F10 mousemelanoma cells with strong pigmentation (upper dashed curve) and weakpigmentation (lower dashed curve), spectra normalized using PA signalsfor the single mouse melanoma cells with strong pigmentation (blackcircles) and weak pigmentation (black squares), and absorption spectrafor pure Hb and HbO₂ (fragments of solid curves in the spectral range630-850 nm).

FIG. 10A is a graph showing the frequencies of circulating mousemelanoma cells (B16F10) detected with label-free PAFC in 50-μm mouse earveins, with a flow velocity of 5 mm/s, in mice with low melaninpigmentation as a function of post-injection time.

FIG. 10B is a graph showing the frequencies of circulating mousemelanoma cells (B16F10) detected with label-free PAFC in 50-μm mouse earveins, with a flow velocity of 5 mm/s, in mice with high melaninpigmentation as a function of post-injection time.

FIG. 11 is a summary of the PA signal rates from single melanoma cellsdetected in a mouse ear lymph microvessel 5 days after tumorinoculation.

FIG. 12 is a summary of the PA signal rates from a single RBC (whitebar) and lymphocytes (black bars) detected by PAFC in the lymph flow ofrat mesentery.

FIG. 13A shows oscilloscope traces of the two-wavelength, time-resolveddetection of PA signals from necrotic lymphocytes labeled with goldnanorods absorbing 639 nm laser pulses.

FIG. 13B shows oscilloscope traces of the two-wavelength, time-resolveddetection of PA signals from apoptotic lymphocytes labeled with goldnanoshells absorbing 865 nm laser pulses.

FIG. 13C shows oscilloscope traces of the two-wavelength, time-resolveddetection of PA signals from live neutrophils labeled with carbonnanotubes absorbing both the 639 nm and the 865 nm laser pulses.

FIG. 14A shows oscilloscope traces of the two-wavelength, time-resolveddetection of PA signals from melanoma cells absorbing 865 nm and 639 nmlaser pulses.

FIG. 14B shows oscilloscope traces of the two-wavelength, time-resolveddetection of PA signals from red blood cells absorbing 865 nm and 639 nmlaser pulses.

FIG. 15 is a summary of the PA signal rates from melanin particlesdetected in a mouse ear lymph microvessel 2 hours after injection.

FIG. 16 is a summary of the PA signal rates from single melanoma cellsdetected in a mouse ear lymph microvessel 4 weeks after tumorinoculation.

FIG. 17 is a summary of the PA signal amplitude generated by quantum dotmarkers as a function of laser fluence.

FIG. 18 is a summary of the PA signal amplitude generated by quantum dotmarkers as a function of the number of laser pulses.

FIG. 19 is a summary of the PA signal amplitudes from a capillary overthe 20 minutes following the injection of magnetic nanoparticles.

FIG. 20 is a summary of the PA signal rates from single melanoma cellsand bacteria cells labeled with magnetic nanoparticles detected in amouse ear capillary 30 minutes after injection.

FIG. 21 is a summary of the PA signal rates from melanoma cells labeledwith magnetic nanoparticles before and after the application of amagnetic field, detected in a mouse ear capillary 20 minutes afterinjection.

FIG. 22 is a summary of the PA signal rates from bacterial cells labeledwith magnetic nanoparticles before and after the application of amagnetic field, detected in a mouse ear capillary 20 minutes afterinjection.

Corresponding reference characters indicate corresponding elements amongthe views of the drawings. The headings used in the figures should notbe interpreted to limit the scope of the claims.

DETAILED DESCRIPTION I. Overview

The present invention is directed to a photoacoustic flow cytometry(PAFC) device 10, as shown in FIG. 2, used to perform non-invasive invivo flow cytometry of at least one type of target object 40 circulatingwithin the vessel of a living organism. The PAFC device 10 of thepresent invention includes a tunable wavelength pulsed laser 20 capableof emitting light energy ranging between wavelengths of about 400 nm andabout 2500 nm. The tunable wavelength pulsed laser source 20 includes apulsed laser 22, and may further include an optical module 26 to convertthe wavelength, pulse rate, or both wavelength and pulse rate emitted bythe pulsed laser 22 to desired values. In addition, the PAFC device 10includes optical elements 30 such as lenses or optic fibers to directthe laser light to the target objects 40. The PAFC device 10 alsoincludes at least one ultrasound transducer 50 to detect photoacousticwaves 42 emitted by the target objects 40. The PAFC device 10 mayoptionally include an amplifier 52, a data recording system 54, and acomputer 56 with stored data analysis software 58.

II. Principle of Operation

The PAFC device 10 is operated by illuminating a circulating targetobject 40 with laser energy pulses 32, thereby inducing the targetobjects 40, for example unlabeled blood cells, to emit a photoacoustic(PA) signal 42. The PA signal 42 is typically in the ultrasoundspectrum, with a range of frequencies between about 20 kHz and about 200MHz. The PA signal 42 emitted by the target objects 40 may result fromthe absorption of laser pulse 32 energy by a variety of mechanismsincluding, but not limited to single photon absorption, two photonabsorption, multi-photon absorption, Coherent Anti-Stokes RamanScattering (CARS), and combinations thereof.

The ultrasound transducer 50 detects the PA signal 42 emitted by thetarget object 40, and the output 60 from the ultrasound transducer 50 isanalyzed using data processing software 58 to identify the presence ofthe target objects 40. In an embodiment, an amplifier 52 may amplify theoutput 60 of the ultrasound transducer 50. In an embodiment, theamplified signal 64 may be stored in a data recording system 54. In anembodiment, the computer 56 may access the stored signal data 64 foranalysis using the data analysis software 58.

Because the ultrasound waves of the PA signals travel freely throughmost biological tissues, the PAFC device 10 may be used to detectcirculating target objects 40 in circulatory and lymphatic vessels asdeep as 15 cm below the external surface of the organism. Further,because the laser power used by the PAFC device 10 is relatively low dueto the efficient absorption of laser light by target objects 40, in vivoPAFC may be conducted for extended time periods with minimal damage toany circulating cells. The PAFC may be used for the continuousmonitoring of circulating cells for the early diagnosis and treatment ofmetastasis, inflammations, sepsis, immunodeficiency disorders, strokes,or heart attacks.

III. Target Objects

The method of the present invention may be used to detect target objects40 circulating in vessels, defined herein as circulatory and lymphaticvessels at a depth between about 10 μm and about 15 cm below the surfaceof the skin and may include capillaries, arterioles, venules, arteries,veins, lymphatic vessels, hyphae, phloem, xylem, and sinuses. Thediameters of the vessels may range between about 10 μm and about 2 cm.The vessels may be located in many different organs and tissues,including, but not limited to skin, lips, eyelid, interdigital membrane,retina, ear, nail pad, scrotum, lymph nodes, brain, breast, prostate,lung, colon, spleen, liver, kidney, pancreas, heart, testicles, ovaries,lungs, uterus, skeletal muscle, smooth muscle, and bladder.

In an embodiment, the at least one laser pulse 32 may be directed to asingle location along one vessel, to two or more locations along asingle vessel simultaneously, to two or more locations along a singlevessel at two or more times, or simultaneously to locations on two ormore vessels.

The method of the present invention may be used on organisms thatpossess cells circulating in vessels or sinuses, from the group oforganisms including mammals, reptiles, birds, amphibians, fish, plants,fungi, mollusks, insects, arachnids, annelids, arthropods, roundworms,and flatworms.

The target objects 40 detected by the method of the present inventionmay be at least one target object 40 including but not limited tounlabeled biological cells, biological cell products, unbound contrastagents, biological cells labeled using contrast agents, and anycombination thereof. The target objects 40 can be unlabeled endogenousor exogenous biological cells or cell products including but not limitedto normal, apoptotic and necrotic red blood cells and white blood cells;aggregated red blood cells or clots; infected cells; inflamed cells;stem cells; dendritic cells; platelets; metastatic cancer cellsresulting from melanoma, leukemia, breast cancer, prostate cancer,ovarian cancer, and testicular cancer; bacteria; viruses; fungal cells;protozoa; microorganisms; pathogens; animal cells; plant cells; andleukocytes activated by various antigens during an inflammatory reactionand combinations thereof.

The target objects 40 that are unlabeled biological cells may possessintrinsic cell-specific markers from the group comprised of hemoglobin(Hb), HbH, HbO₂, metHb, HbCN, HbS, HbCO, HbChr, myoglobins, melanin,cytochromes, bilirubin, catalase, porphyrins, chlorophylls, flavins,carotenoids, phytochromes, psoralens and combinations thereof.

The target objects 40 may also be biological cell products, includingbut not limited to products resulting from cell metabolism or apoptosis,cytokines or chemokines associated with the response of immune systemcells to infection, exotoxins and endotoxins produced during infections,specific gene markers of cancer cells such as tyrosinase mRNA, p97,MelanA/Mart1 produced by melanoma cells, PSA produced by prostatecancer, and cytokeratins produced by breast carcinoma.

The target objects 40 may also be contrast agents from the groupcomprised of indocyanine green dye, melanin, fluoroscein isothiocyanate(FITC) dye, Evans blue dye, Lymphazurin dye, trypan blue dye, methyleneblue dye, propidium iodide, Annexin, Oreg. Green, C3, Cy5, Cy7, NeutralRed dye, phenol red dye, AlexaFluor dye, Texas red dye, goldnanospheres, gold nanoshells, gold nanorods, gold cages, carbonnanoparticles, perfluorocarbon nanoparticles, carbon nanotubes, carbonnanohorns, magnetic nanoparticles, quantum dots, binary gold-carbonnanotube nanoparticles, multilayer nanoparticles, clusterednanoparticles, liposomes, liposomes loaded with contrast dyes, liposomesloaded with nanoparticles, micelles, micelles loaded with contrast dyes,micelles loaded with nanoparticles, microbubbles, microbubbles loadedwith contrast dyes, microbubbles loaded with nanoparticles, dendrimers,aquasomes, lipopolyplexes, nanoemulsions, polymeric nanoparticles, andcombinations thereof.

The multilayer nanoparticles used as contrast agents for the targetobjects 40 may include two or more layers of materials with optical,thermal, and acoustic properties that enhance the PA signals 42 emittedby the target objects 40. Non-limiting examples of the effects of themultilayered nanoparticles on the PA pulses 42 emitted by the targetobjects 40 labeled with the multilayered nanoparticles include enhancingabsorption of the laser pulse energy, increasing thermal relaxationtime, increasing acoustic relaxation time, increasing the coefficient ofthermal expansion, decreasing the coefficient of thermal diffusion,decreasing the local speed of sound near the target object 40,decreasing the threshold of bubble formation of the target object 40 andcombinations thereof.

The target objects 40 may also be labeled living cells from the listabove, marked with molecular markers and tags comprised of contrastagents selected from the list above. The molecular markers or tags maybe attached to the cells without modification, or the contrast agentsmay be functionalized for binding to the cells using molecules includingbut not limited to antibodies, proteins, folates, ligands for specificcell receptors, receptors, peptides, viramines, wheat germ agglutinin,and combinations thereof. The ligands may include but not limited toligands specific to folate, epithelial cell adhesion molecule (Ep-CAM),Hep-2, PAR, CD44, epidermal growth factor receptor (EGFR), as well asreceptors of cancer cells, stem cells receptors, protein A receptors ofStaphylococcus aureus, chitin receptors of yeasts, ligands specific toblood or lymphatic cell endothelial markers, as well as polysaccharideand siderophore receptors of bacteria.

Exogenous target objects 40 such as unbound contrast agents andexogenous unlabeled biological cells may be introduced into thecirculatory or lymphatic vessels of the organism perenterally, orally,intradermally, subcutaneously, or by intravenous or intraperitonealadministration.

The target objects 40 may be concentrated near the desired area ofdetection using a variety of techniques. For target objects 40possessing a larger diameter than the surrounding cells, the lumen ofthe vessel in which the target cells 40 are to be detected may bereduced in cross-sectional area using gentle mechanical pressure on thetissue surrounding the vessel, thereby retaining the larger diametertarget cells 40, while allowing the surrounding cells with smallerdiameter than the target objects 40 to flow away unimpeded from thedesired area of detection. Alternatively, the target objects 40,regardless of object diameter, may be marked with magnetically activetags or markers, and the target objects 40 may be held in place by themagnetic forces exerted by a magnet with a magnetic field strength of atleast seven Tesla, placed near the desired area of detection.

To further increase the contrast between PA signals 42 or originatingfrom the target objects 40 and the background PA signals fromsurrounding cells and tissues, a variety of approaches may be used. Theorganism may be exposed to hyperoxic or hypoxic atmospheric conditionsto induce different levels of oxygenation, which in turn alters thelight absorption properties of the red blood cells. The osmolarity ofthe vessel flow may be altered by injecting hypertonic or hypotonicsolutions into the desired vessel, thereby causing physical swelling orshrinking of surrounding cells, and further altering the lightabsorption characteristics of the surrounding cells. The hematocrit ofthe vessel flow may be altered by the injection of a diluting solutioninto the vessel flow, thereby reducing the density of surrounding cellsin the vessel, and the resulting light absorption characteristics of thesurrounding cells.

IV. Tunable Wavelength Pulsed Laser

The PAFC device 10 includes a tunable wavelength pulsed laser 20 whichgenerates light energy at laser pulse widths ranging between about 0.1ps and about 1000 ns, at least one wavelength ranging between about 400nm and about 2500 nm, laser pulse rates ranging between about 1 Hz andabout 500,000 Hz, and laser pulse energy fluences ranging between about0.1 mJ/cm² and about 10 J/cm². A variety of tunable wavelength pulsedlasers 20 may be used to produce the laser pulse 28 so long as the laserenergy is delivered in such a way that minimal damage occurs to anytarget objects 40 or surrounding cells or tissues that are illuminatedby the device 10. The laser pulses of at least one wavelength may bedelivered by a single tunable wavelength pulsed laser 20 or by an arrayof tunable wavelengths pulsed lasers 20, with each laser source 20delivering laser pulses 28 of different wavelengths.

Several characteristics of the tunable wavelength pulsed laser 20strongly influence the performance of the device 10, including thewavelength of light emitted by the laser (λ), the diameter of the beamemitted by the laser, the duration of the light pulses emitted by thelaser (t_(P)), the laser pulse repetition rate (f), and the laserfluence defined herein as the amount of energy emitted per squarecentimeter by the tunable wavelength pulsed laser 20.

A) Wavelength of Laser Pulse

The wavelengths of the laser pulses 32 may be selected to optimize thecontrast of a single type of target object 40, or to optimize thecontrast of two or more types of target objects 40. Typically, but notnecessarily, to maximize the sensitivity and resolution of the PAFCdevice 10, the one or more wavelengths of laser pulses 32 should beoptimized to enhance the contrast between the induced PA signals fromthe target objects 40 and the PA signals emitted by any surroundingcells. Thus, for target objects 40 that are unlabelled cells, thewavelength is selected in a range of the spectrum near the maximumabsorbance of the unlabelled cells and as far as possible from themaximum absorbance of surrounding cells. If the target objects 40 areunbound contrast agents or cells that are labeled with contrast agents,the wavelength of the laser pulse 32 must fall within the range ofwavelengths that are maximally absorbed by the contrast agents. As such,the present invention uses wavelengths of electromagnetic radiationemitted at wavelengths ranging between about 10 Å and about 1 cm. In anembodiment, the PAFC device 10 uses wavelengths of light emitted in thenear-infrared spectrum ranging between about 400 nm and about 2500 nm.

In an embodiment, the PAFC device 10 may use diode lasers that generatelaser pulses 32 with wavelengths ranging between about 640 nm and about680 nm, between about 790 nm and about 830 nm, or between about 880 nmand about 930 nm. In an embodiment, the PAFC device 10 may use laserpulses or modulated continuous radiation in the x-ray spectrum (1-10 Å),the terahertz spectra (20-1000 μm) or the microwave spectra (0.5 mm-3cm).

B) Laser Beam Dimensions

The size and spacing of the target objects 40 typically, but notnecessarily, determine the laser beam's 32 cross-sectional shape anddimensions of the laser beam 32. To distinguish closely spaced targetobjects 40 in circulation, the minimum laser beam 32 dimension should beno smaller than the diameter of the target objects 40. For the sizerange of potential target objects 40 such as unbound nanoparticles,bacteria, blood cells, or metastatic cells, the minimum laser beam 32dimension ranges between about 1 μm and about 20 μm.

The laser beams 32 may have a circular cross-section, with diameterscomparable to the blood or lymph vessel diameters, for detecting raretarget objects 40 that may be target cells separated by distances of atleast 100 μm in circulation. For the detection of many closely-spacedtarget objects 40 in larger vessels, the laser beam 32 may be adjustedusing known optical methods to an elliptical cross-sectional shape, withthe long axis of the ellipse set to be the diameter of the largervessel, and the short axis of the ellipse set to be the diameter of thecell. Thus, the laser beam 32 dimensions may range between about 1 μmand about 150 μm, in either a circular or an elliptical cross-sectionalshape, depending on the relative rarity of the target objects 40, thedimensions of the target objects 40, and the diameter of the vessel inwhich the target objects 40 are detected.

Although any device which supplies light energy with the abovecharacteristics may be used, the tunable wavelength pulsed laser 20 mayinclude but is not limited to a pulsed or modulated continuous laser 22optically connected to an optical module 32. The laser source 22 mayinclude but is not limited to gas lasers, chemical lasers, excimerlasers, solid state lasers, fiber-hosted lasers, semiconductor (diode)lasers, dye lasers, and free electron lasers. The optical module 32converts the wavelength of light emitted by the pulsed laser 26 to atleast one different wavelength used for in vivo PAFC of the targetobjects 40 as specified above, typically within the visible and NIRspectral ranges. The optical module 32 can be any device capable ofconverting the laser wavelength or pulse rate to the desired wavelengthor pulse rate using linear or non-linear optical effects, including butnot limited to optical parametric oscillators, optical crystals,etalons, monochromatic filters, distributed Bragg reflector structures,Lyot filters, Raman shifters, or combinations thereof.

C) Laser Pulse Duration

Typically, but not necessarily, to generate the maximum PA signal 42with optimal conversion of light energy into acoustic energy, the laserpulse 32 duration t_(P) is predetermined to fall below a predeterminedacoustic confinement criteria in order to minimize the mechanicalstresses acting on the target object 40. This criterion may be expressedin equation form:t _(P)≤τ_(A)=2R/c _(s)  Eqn. (1)where τ_(A) is the transit time of the acoustic wave traveling throughthe target object 40, R is the radius of the target object 40, and c_(s)is the speed of sound inside the target object 40. Assuming a targetobject 40 diameter ranging between about 0.5 μm and about 15 μm fortypical cells such as bacteria, blood, and metastatic cells, andassuming that the speed of sound inside the target objects 40 isapproximately the same as the speed of sound in water (1.5×10⁵ cm/sec),then the range of the pulse durations required for detecting mostpotential target objects 40 ranges between about 0.7 ns and about 20 ns.Smaller bacterial cells may use pulse durations near the lower end ofthe range specified above, and larger metastatic cells may use pulsedurations near the upper end of this range. Extremely small targetobjects 40 such as individual nanoparticles may require pulse durationsof about 10⁻⁴ ns. The device 10 typically uses a pulse duration rangingbetween about 1 ns and about 20 ns, depending on the size of targetobjects 40.D) Laser Pulse Repetition Rate

To accurately identify and discriminate between numerous circulatingtarget objects 40 in relatively fast flow conditions, the pulsed laser22 must typically, but not necessarily, have a pulse repetition ratethat is sufficiently rapid to ensure that only one target object 40 isilluminated per pulse. In equation form, the laser pulse repetition rate(f) must fulfill the following predetermined criterion:f≥(V _(F))/D  Eqn. (2)where (V_(F)) is the flow velocity, and D is the diameter of the targetobject 40. In small mammal blood microvessels, flow velocities typicallyrange from 1 mm/sec (capillary) to 10 mm/sec (arterioles). For a celldiameter of 20 μm, the pulse repetition rate may range between about 50Hz and about 500 Hz. To detect smaller target objects 40, or targetobjects 40 in faster flowing vessels, the pulse repetition rate shouldbe near higher end of this range. A high laser pulse repetition rate mayalso enhance the sensitivity of the device 10 during multi-pulse laserexposures because signal-to-noise ratio, which limits the sensitivity ofthe device 10, is proportional to the square root of the number of laserpulses. In an embodiment, the PAFC device 10 of the present inventionuses a pulse repetition rate ranging between about 1 Hz and about500,000 Hz.

In addition, when the one or more target objects 40 are illuminated bylaser pulses of two or more different wavelengths, the time delaybetween the laser pulse 32 of one wavelength and the subsequent laserpulse 32 of a different wavelength should be sufficiently short so as toensure that the two or more laser pulses illuminate the same targetobject 40. Further, the laser pulses 28 should have a time delay thatfurther ensures that the second laser pulse 32 does not reach the targetobject 40 prior to detection of the PA signal 42 induced by the firstlaser pulse 32. For the range of distances at which the PAFC device 10detects target objects 40, the time delay between laser pulses 32 rangesbetween about 0.1 μs and about 100 μs.

E) Laser Fluence

The laser fluence of the PAFC device 10, defined herein as the energylevel of the laser pulse 32, should not exceed the ANSI safety standard,which depends on the laser's wavelength, and ranges between about 30mJ/cm² and about 100 mJ/cm² in the NIR spectral region emitted by thelaser 20 in an embodiment. In addition, the laser fluence should notexceed the thresholds at which significant cell photodamage may occur.The PAFC device 10 develops a laser fluence ranging between about 0.1mJ/cm² and about 1000 J/cm², depending on factors such as the size andtype of target objects 40, the depth of the vessel in which the targetobjects 40 are to be detected, and the density of the cells surroundingthe vessel. When lower laser fluences are used, the resulting PA signals42 are proportionally weaker in amplitude, and a more sensitive acousticdetection system 10 with signal acquisition properties that areoptimized for the location and frequency of the PA signals 42 may beused to provide reliable detection of PA signals 42 from the targetobjects 40.

For PAFC measurements on vessels in deeper tissues, the laser pulses 32may be delivered non-invasively at higher laser fluence from outside theorganism. To avoid potential damage to any tissues located between thepulsed laser 20 and the target objects 40 resulting from higher energylaser pulses 32 or extended periods of PAFC measurement, the tissues maybe cooled using methods broadly used in dermatological laserapplications including but not limited to spray cooling, contactcooling, skin cooling with forced cooled air or liquid flow, anoptically transparent cooling device attached to skin and cooled usingcirculating cooled water or electrical effects, and combinationsthereof.

In an embodiment, laser pulses 32 may be delivered using a fiber opticcable placed in close vicinity of the target objects 40 using aminimally invasive needle delivery device. The laser pulses 32 may bedelivered directly to the desired vessel using an optic fiber cablemounted in a catheter. The laser pulses 32 may be delivered byfiber-chip-based catheters inserted directly into the desired vessel.The target objects 40 may be detected by shunting circulating targetobjects 40 through artificial circulatory bypass tubes similar to thoseused in hemodialysis, that are transparent to laser light in the visibleor NIR spectra, through a hemodialysis system, or through similarbioengineering devices known in the art.

V. Optics

Optics 30 are operatively connected to the output 28 of the tunablewavelength pulsed laser 20, and functions to deliver the laser pulses 32to the target objects 40 or other desired area of illumination. Theoptics 30 are selected to deliver the laser pulses 32 to the targetobjects 40 with a beam dimension ranging between about 1 μm and about 40μm, with a circular or elliptical cross-sectional geometry, as discussedabove. The optics 30 may include but are not limited to conventionallenses, mirrors, optics fibers, and combinations thereof, so long as theshape and diameter of the beam may be controlled.

VI. Ultrasound Transducers

The ultrasound transducers 50 are pressure sensors that are placed inacoustical contact with the target objects 40 at a distance of up to 15cm from the target objects 40. The ultrasound transducers 50 convert thelaser-induced PA signals 42 received from the target objects 40 intovoltage fluctuations that are subsequently amplified, digitized, stored,and/or analyzed. The ultrasonic transducers 50 are selected for theiroptimal sensitivity to the PA signals 42 emitted by the target objects40. The ultrasonic transducers 50 typically have a sample rate rangingbetween about 10 kHz and about 100 MHz. Non-limiting examples ofultrasonic transducers so include, unfocused ultrasound transducers;focused ultrasound transducers with conventional and cylindrical focusedlengths between about 2 mm and about 500 mm; and customized resonanceultrasound transducers. In an embodiment, the ultrasonic transducer 50and pulsed laser 22 may have a confocal configuration, in which thetransducer 50 may have a ring geometry with the pulsed laser 22 andassociated optics 30 passing through the center of the ring of thetransducer 50. In an embodiment, the PAFC device 10 may include one ormore ultrasonic transducers 50.

The efficiency of acoustic matching of the ultrasound transducer 50 andthe tissue of the organism may be enhanced by the application of anacoustically transparent liquid, such as glycerol, between the skin andthe receiving surface of the ultrasound transducer 50. Any liquid may beplaced between the skin and the receiving surface of the ultrasoundtransducer 50, so long as the liquid efficiently transmits ultrasoundpressure waves. In addition, the acoustically transparent liquid shouldalso transmit laser pulses in the visible and NIR spectra with minimalscattering of the laser pulse energy, if the liquid is located in thepath of the laser pulse 32.

VII. Amplifier

An optional amplifier 52 may be electrically connected to the voltageoutput 60 of the ultrasonic transducer 50, should the PA signal 42received from the target objects 40 prove to be too weak to analyzeaccurately. The amplifier 52 is selected for sensitivity andresponsiveness to the PA signals 42 measured by the one or moreultrasonic transducers 50. The amplifier 52 is selected to possess a lowfrequency boundary between about 10 kHz and about 200 kHz, a highfrequency boundary between about 1 MHz and about 100 MHz, and aresonance bandwidth between about 0.3 MHz and about 5 MHz. Any amplifier52 can be used so long as it possesses the frequency response andresonance bandwidth described above.

VIII. Data Recording System

A data recording system 54 may be connected to the voltage output 60 ofthe ultrasonic transducer 50, or alternatively, the amplified output 62of the ultrasonic transducer 50. Any digital or analog device capable ofacquiring and storing incoming voltage data at frequencies as high as 50MHz may be used. Non-limiting examples of the data recording system 54include a boxcar averager device, a video camera recording the displayof an oscilloscope electrically connected to receive the output of theultrasonic transducer 50 or the amplifier 52, and combinations thereof.A boxcar averager device averages the output of several successive PAsignals 42 to optimize the accuracy of the PAFC device 10. Theoscilloscope display may be run at low speed to acquire the counts ofmany target objects 40 passing the PAFC device 10 in close succession,or the oscilloscope display may be run at high speed to discern detailedcharacteristics of the PA signal 42 such as wave magnitude or wave shapeused to discriminate the target object 40 from surrounding cells. Thedata recording system 54 may include a still camera may be used torecord the display an oscilloscope connected to the output of theultrasonic transducer 50 or the amplifier 52.

IX. Data Analysis Software

The data analysis software 58 may access the stored data 64 from thedata storage device 54, the voltage output 60 from the ultrasonictransducer 50, the voltage output 62 from the amplifier 52, orcombinations thereof. The data analysis software 58 may also function asan amplifier 52, a data storage device 54, and combinations thereof. Anydata analysis software 58 capable of processing data that fluctuates atfrequencies ranging between about 20 Hz and 200 MHz may be used,including but not limited to user-written software, or commerciallyavailable analysis software. Non-limiting examples of commerciallyavailable analysis software includes Matlab (The MathWorks, Inc., USA),Mathematica (Wolfram Research, Inc., USA), Labview (National Instrument,USA), Avisoft (Avisoft Bioacoustics, Germany), and TomoView (Olympus NDTInc., USA).

X. Combined Photoacoustic/Photothermal/Fluorescent Flow CytometrySystems

The photoacoustic flow cytometry (PAFC) device 10 may include additionalelements including, but not limited to photodetectors, additional lasersand optics, and additional analysis software associated with other invivo flow cytometry methods that detect the target objects using theconventional and Raman scattering of the laser pulses by the targetobjects, photothermal effects induced by laser pulses on the targetobjects, and the fluorescence of the target objects induced by absorbedlaser pulses. In an embodiment, the device 10 may be configured tosimultaneously detect cells using photoacoustic methods, photothermalmethods, light scattered by target objects, induced fluorescence oftarget objects, and any combination thereof.

XI. Method of In Vivo Flow Cytometry

The present invention further provides a method of in vivo flowcytometry using laser-excited photoacoustic pulses 42 emitted by atleast one type of target object 40 circulating in the vessels of anorganism. The method includes pulsing the target objects 40 movingthrough the vessels with at least one pulse of laser energy 32 at one ormore wavelengths. As described above, the target objects 40 absorb theat least one pulse of laser energy 32 and emit at least one ultrasoundphotoacoustic pulse 42. The photoacoustic pulses 42 are detected by atleast one ultrasound transducer 50, as described above. Thephotoacoustic pulses 42 are analyzed to determine at least onecharacteristic of the detected target objects 40. The characteristics ofthe detected target objects 40 may include, but are not limited to thetype of target objects 40, the quantity of target objects 40, theconcentration of target objects 40, the flow speed of the target objects40, the total blood volume, and combinations thereof.

The flow speed of the target objects 40 may be based on analysis of thewidth of the PA signal 42 emitted by a target object 40, the time delaybetween two PA signals 42 measured at two locations separated by a knowndistance, or the frequency shift of a PA signal 42. The totalcirculating blood or lymph volume may be estimated using the degree ofdilution of one or more absorbing dyes, or blood cells extracted fromthe organism, labeled using the marking compounds discussed above, andreintroduced into the vessels of the organism.

A) PA Signal Patterns

Signature PA signal patterns associated with each type of target object40, include but are not limited to signal shape, frequency spectrum,amplitude, phase, and time delay between the one or more laser pulses 32and the received PA signal 42. The PA signal patterns may bediscriminated between PA signals 42 received from target objects 40 andbackground PA signals received from surrounding cells, blood andlymphatic vessel walls, and tissues. Further, the blood or lymph flowvelocity may be determined using PA signal patterns including but notlimited to the PA signal duration, the PA frequency shift, or the timedelay between two PA signals 42 produced by a single target object 40pulsed by two distinct laser pulses 32 applied at a known separationdistance.

Different target object 40 types possess unique combinations of pigmentsand sub-cellular structures that absorb laser pulses 32 and emit PAsignals 42 differently. Each type of target object 40 may bediscriminated by its distinctive PA signature, as well as the particularwavelengths of laser pulses 32 used to elicit the PA signals 42 fromtarget objects 40 without need for labeling. The contrast of the targetobjects 40 may be enhanced using contrast agents bound to the targetobjects 40 as described above.

The contrast of the PA signal patterns of the target objects 40 relativeto surrounding cells and tissues are typically based on PA signal 42amplitudes from the target objects 40 that are significantly higher thanamplitudes of the PA signals 42 amplitudes from the surrounding cellsand tissues. For example, aggregations of red blood cells in circulationmay be detected using time-resolved monitoring of dynamic increases ofPA signal amplitudes due to the higher local absorption of laser pulses32 that result in higher amplitude PA signals 42 from the red blood cellaggregates relative to surrounding cells and tissues.

The PA signal amplitude emitted by target objects 40 may also besignificantly lower than the PA signal 42 amplitudes from thesurrounding cells and tissues. For example, circulating blood clots maybe detected through the time-resolved monitoring of dynamic decreases ofthe PA signal amplitude, due to the attenuated PA signal amplitudeemitted by blood clots relative to the PA signal amplitude emitted byred blood cells. The decreased PA signal amplitude emitted by bloodclots is due to the lower light absorption of platelets (the dominantcomponent of blood clots) relative to the light absorption of red bloodcells (the dominant cell type overall in typical blood flows) in thevisible and near-infrared spectral range. Whether the PA signal 42 ofthe target objects 40 is significantly higher or significantly lowerthan the PA signal 42 from surrounding cells and tissues, sufficientcontrast must exist to accurately detect the PA signals of the targetcells 40.

B) Detection of Target Objects in Lymph Vessels

For the detection of target objects 40 in the lymph vessels, targetobjects 40 are illuminated while passing between the leaves of a valvein a lymphatic vessel. The restricted flow through the valve of thelymphatic vessel facilitates the detection and identification ofindividual target objects 40. The phasic contractions of lymph vesselsnaturally concentrate the flow of target objects 40 near the center ofthe lymph vessel, and the lymph valves act as natural nozzles to providethe positioning of target objects 40 in single file with minimum radialfluctuation. To exclude detecting the same target objects 40 duringtheir retrograde motion, as is typical in lymphatic vessels, the timingof the laser pulses 32 may be adjusted to synchronize with the phasicrhythms of the lymphatic flow. In addition, detection of forward movingtarget objects 40 may be achieved by synchronizing the laser pulses 32with the motion of the lymphatic vessel wall or lymphatic valves thattypically occurs during forward flow of lymph. The motion of the lymphvessel wall or lymphatic valves may be sensed using an additional pilotlaser that produces signals that may be used to trigger the PAFC laserpulses 32.

The sensitivity of the detection of target objects 40 in lymph vesselsmaybe enhanced by creating a higher rate of lymph flow through thelymphatic valve by inducing the contraction of the upstream lymphangion,defined herein as the lymph vessel between two lymphatic valves. Forexample, the lymphangion may be induced to contract by exposure to apulse of laser light 32 at a wavelength ranging between about 400 nm andabout 950 nm, applied prior to the laser pulses 32 used to induce PAsignals 42 from the target objects 40.

XII. Method of In Vivo Detection of Circulating Melanoma Cells

The present invention further provides a method for non-invasivedetection of circulating unlabelled metastatic melanoma cells. Themethod includes pulsing at least one circulating metastatic melanomacell with at least one pulse of NIR laser energy 32 at least onewavelength ranging between about 650 nm and about 950 nm and a laserfluence ranging between about 20 mJ/cm² and about 100 mJ/cm². Thecirculating unlabelled metastatic melanoma cells absorb the at least onelaser pulse 32 and emit at least one ultrasound photoacoustic pulse 42,that is detected by at least one ultrasound transducer 50. The detectedphotoacoustic pulse 42 is analyzed to detect the presence of anymetastatic melanoma cells in circulation.

Unlabelled metastatic melanoma cells in particular may be detected inthe circulatory or lymphatic system of the organism using the method ofthe present invention. Without being bound to any particular theory, thelarge stores of melanin characteristic of melanoma cells readily absorblight at a wavelength of approximately 850 nm and emit strongphotoacoustic ultrasound signals 42. Using the device 10 of the presentinvention with a tunable wavelength pulsed laser 20 with a pulsewavelength of about 850 nm, a laser fluence of about 35 mJ/cm², and alaser pulse duration of about 8 ns, circulating unlabelled metastaticmelanoma cells may be detected with a resolution of at least 1 melanomacell per 10¹⁰ cells.

XIII. Method for Selectively Destroying Circulating Cells In Vivo

The present invention provides a method of selectively destroying targetobjects 40 including but not limited to metastatic cancer cells usinglaser pulses 32 with a higher laser fluence than is typically used bythe PAFC device 10. The method includes detecting the target objects 40circulating in the vessels. The detection of a target object 40 triggersa pulse of laser energy 32 that is delivered to the detected targetobject 40 at a wavelength and fluence sufficient to cause thedestruction of the detected target objects 40. The method may includemonitoring the frequency of detection of target objects 40 circulatingthrough the vessels, and terminating the method when the frequency ofdetection of the target objects 40 falls below a threshold level. Themethod of destroying target objects 40 may be terminated when thefrequency of detection of target objects 40 falls below about 10⁻³target objects/min and about 10² target objects/min.

The target objects 40 may be detected using a method of in vivo flowcytometry using laser-excited photoacoustic waves 42 emitted by thetarget objects 40.

Because the absorption of target objects 40 are much higher thansurrounding cells and tissue, the laser fluence may be increased beyondthe level normally used for PAFC to levels that selectively damage thetarget objects 40 without harming the surrounding cells and tissues.Target objects 40 that are selectively destroyed using the method of thepresent invention include but are not limited to tumor cells, bacteria,viruses, clots, thromboses, plaques, and combinations thereof. Thisapproach may be used for the selective destruction of target objects 40circulating in blood vessels or lymph vessels. The PAFC methods of thepresent invention may be used to detect the target objects 40, triggerthe high energy laser pulse 32 used to destroy the target objects 40,and detect the subsequent decrease in the target objects 40, therebyguiding the cell destruction at the single cell level.

EXAMPLES

The following examples illustrate the invention.

Example 1 In Vitro Photothermal (PT) Imaging was Used to Determine theEffect of Laser Energy Levels on Laser-Induced Cell Damage to BloodCells and Subsequent Cell Viability

To determine whether the laser pulses using in in vivo flow cytometrycaused any significant damage to cells or tissues of the organism, thefollowing experiment was conducted. The laser-induced damage thresholdof single cells was evaluated as a function of the pumped-laser energylevels at a range of wavelengths using established methods (Zharov andLapotko 2005, Lapotko and Zharov 2005). In vitro measurements ofspecific changes in photothermal (PT) images and PT responses fromindividual cells were used to determine cell damage. During the PTimaging, individual cells were illuminated with a pulse of laser lightat a specified energy level and wavelength. After absorbing the energyof the laser pulse, the short-term temperature of the cell increased byas much as 5° C. The laser-induced temperature-dependent refractiveheterogeneity in the vicinity of cells caused defocusing of a collinearHe—Ne laser probe beam (model 117A; Spectra-Physics, Inc.; 633 nm, 1.4mW) that illuminated the cell immediately after the initial laser pulse.This defocusing caused a subsequent reduction in the beam's intensity atits center, which was detected with a photodiode (C5658; HamamatsuCorp.) through a 0.5-mm-diameter pinhole.

PT measurements were performed in vitro using mouse blood cells insuspension on conventional microscope slides. To simulate blood flowconditions, a flow module fitted with a syringe pump-driven system (KDScientific, Inc.) was used with glass microtubes of different diametersin the range of 30 μm to 4 mm that provided flow velocities of 1-10cm/sec, which were representative of the diameters and flow rates ofanimal microvessels.

Individual cells flowing through the glass microtubes were exposed to an8 ns burst of laser light in a 20-μm circular or elongated beam at avariety of wavelengths ranging between 420 nm and 2300 nm. At eachwavelength of the initial laser pulse, the laser fluence, defined as theenergy contained in the laser beam, was varied between 0.1 mJ/cm² and1000 J/cm². Damage to the cells was determined by assessing the changesin the PT imaging response of cells to laser pulses of increasingfluence. In addition, cell viability after exposure to laser energy wasassessed using a conventional trypan blue exclusion assay. Cellulardamage was quantified as ED50, the level of laser fluence at which 50%of the measured cells sustained photodamage in vitro. The ED50 valuesmeasured for rat red blood cells (RBC), white blood cells (WBC) and K562blast cells using laser pulses in the visible light spectrum aresummarized in Table 1. The ED50 values measured for rat red blood cells(RBC) and white blood cells (WBC) using laser pulses in thenear-infrared (NIR) light spectrum are summarized in Table 2.

TABLE 1 Photodamage thresholds for single rat blood cells in the visiblelight spectrum. Wavelength of Photodamage threshold ED50 (J/cm²) laserpulse Rat K562 (nm) Rat RBC Rat WBC blast cell 417 1.5 12 36 555 5 42 90

TABLE 2 Photodamage thresholds for single rat blood cells in near-IRspectral range. Wavelength of Photodamage threshold laser pulse ED50(J/cm²) (nm) Rat RBCs Rat WBCs 740 6.9 21.7 760 6.8 780 17.7 152 80017.5 219 820 28.0 251 840 43.5 860 43.8 730 880 76.5 900 69.4 920 77.7357 960 33.5 48.8

In the visible spectral range, the relatively strong light-absorbingRBCs sustained cell damage at much lower intensities of laser energy,resulting in ED50 values that were about an order of magnitude lowerthan the ED50 values measured for WBC or K562 blasts. In the NIRspectral range, where most cells, including RBC, have minimalabsorption, cells did not sustain damage until much higher laser energylevels compared to the energy levels at which cellular damage occurredto cells exposed to laser energy in the visible spectrum. The damagethresholds (ED50) for RBCs and WBCs in the spectral range of 860-920 nmwere more than one order magnitude higher compared to those in thevisible spectrum as shown in Tables 1 and 2.

The results of this experiment established the levels of laser energy atwhich laser-induced cellular damage may occur. In the NIR spectrum, inwhich cells exhibited the strongest photoacoustic effects, the damagethresholds are several orders of magnitude above the maximum safetylevel of approximately 20-100 mJ/cm² set by ANSI safety standards. Thus,photoacoustic flow cytometry may be performed in vivo with little riskof cell or tissue damage.

Example 2 Prototype In Vivo Photoacoustic Flow Cytometry System Used toDetect Contrast Dye Circulating in Mice

The following experiment was conducted to demonstrate the feasibility ofin vivo photoacoustic flow cytometry (PAFC) for real-time, quantitativemonitoring in the blood circulation of a conventional contrast agent,Lymphazurin. In this experiment, a prototype PAFC system was used todetect Lymphazurin circulating in the blood vessels of a mouse ear.

The prototype PAFC system was built on the platform of an Olympus BX51microscope (Olympus America, Inc.) and a tunable optical parametricoscillator (OPO) pumped by a Nd:YAG laser (Lotis Ltd., Minsk, Belarus).The general layout of the PAFC system is shown in FIG. 2. Laser pulseshad an 8 ns pulse width, a regular repetition rate of 10 Hz with theability to provide short-term pulses at 50 Hz, and a wavelength in therange of 420-2,300 nm. Laser energy was directed to the blood vesselsusing a conventional lens, or an optical fiber. PA waves emitted by thecells were detected by ultrasound transducers (unfocused Panametricsmodel XMS-310, 10 MHz; focused cylindrical Panametrics model V312-SM, 10MHz, focused lengths of 6 mm, 12 mm, and 55 mm; and customized resonancetransducers), and the ultrasound transducer outputs were conditioned byan amplifier (Panametrics model 5662, bandwidth 50 kHz-5 MHz;Panametrics model 5678, bandwidth 50 kHz-40 MHz; customized amplifierswith adjustable high and low frequency boundaries in the range to 50-200KHz and 1-20 MHz, respectively; resonance bandwidth of 0.3-1.0 MHz). Theamplifier output signals were recorded with a Boxcar data acquisitionsystem (Stanford Research Systems, Inc.) and a Tektronix TDS 3032Boscilloscope, and were analyzed using standard and customized software.The Boxcar data acquisition technique provided averaging of the PA wavesfrom cells in the blood vessels, and discriminated the PA waves frombackground signals from surrounding tissue on the basis of thedifference in time delays between the two signals. The signals from theoscilloscope screen were recorded with a digital camera (Sony, Inc.) andvideo camera (JVC, Inc.).

A high-speed computer (Dell Precision 690 workstation with a quadcoreprocessor, 4 GB of RAM and Windows Vista 64 bit operating system) anddigitizer (National Instruments PCI-5124 high speed digitizer) were usedto acquire the PA signal data from the PAFC device. National Instrumentssoftware (Labview Version 8.5 and NI Scope Version 3.4) was used tocontrol the digitizer and create a data logging user interface. Thehardware and supporting program were capable of collecting data at arate of 200 megasamples per second, corresponding to a time resolutionof 5 ns.

A laser beam with a circular cross section and a diameter ofapproximately 50 μm, a wavelength of 650 nm, and a fluence of 35 mJ/cm²was used to illuminate the flow in the blood vessels. The 650 nmwavelength used was near the wavelength of maximum absorption ofLymphazurin, the contrast dye used in this experiment, and wellseparated from the wavelengths of maximum absorption of other bloodcomponents. Navigation of the laser beams was controlled withtransmission digital microscopy (TDM) at a resolution of approximately300 nm using a Cascade 650 CCD camera (Photometrics).

All in vivo experiments described below were performed using a nudemouse ear model. PAFC detection was performed using relativelytransparent, 270 μm thick mouse ears with well-distinguished bloodmicrovessels. The ear blood vessels examined were located at a depth of30-100 μm, had diameters in the range of 30-50 μm, and blood velocitiesof 1-5 mm/sec. After undergoing anesthesia using ketamine/xylazine at adosage of 50/10 mg/kg, each mouse was placed on a customized heatedmicroscope stage, together with a topical application of warm water,which provided acoustic matching between the transducer and mouse ear.

The contrast dye used for the experiments described below wasLymphazurin, a contrast agent commonly used for the delineation oflymphatic vessels. A 1% solution of Lymphazurin (Isosulfan Blue) waspurchased from Ben Venue Labs Inc., USA.

After anaesthetizing each mouse and placing the mouse on the microscopestage as described above, 200 μl of a 1% aqueous solution of Lymphazurinwas injected into the tail vein of the mouse.

PAFC measurements of the circulating dye were performed at a laser pulsewavelength of 650 nm. FIG. 3 shows oscilloscope traces of PAFC signalsfrom the blood vessels and surrounding tissues in the rat ear before andafter injection with Lymphazurin. Prior to injection, the maximum 240 mVPA signals from blood vessels, shown in FIG. 3A, were approximately 1.5times higher than the 160 mV PA background signals from surroundingtissue, shown in FIG. 3B. Maximum PA signals from the blood vessel afterdye administration, shown in FIG. 3C, increased approximately three-foldover pre-injection levels. The PA signals from tissue around vesselsafter dye injections, shown in FIG. 3D, gradually increasedapproximately 2.5-fold over pre-injection levels during the first 15-20minutes, and then remained relatively constant for the next 1-1.5 hours,probably due to the passage of the Lymphazurin out of the blood vesselsand into nearby lymphatic vessels.

FIG. 4 summarizes the maximum PAFC signals from Lymphazurin compared tobackground PAFC signals from the blood vessels, observed for one hourafter the injection of Lymphazurin. As shown in FIG. 4, continuousmonitoring of PA signals from the ear blood microvessels revealed arapid appearance of Lymphazurin in the blood flow within a few minutesafter injection, followed by clearance of Lymphazurin from the bloodover the next 50 minutes.

The results of this experiment demonstrated that the prototype PAFCsystem exhibited sufficient sensitivity to detect the presence ofultrasonic contrast dyes in circulation.

Example 3 Prototype In Vivo Photoacoustic Flow Cytometry System Used toDetect Nanoparticles Circulating in Rats

To demonstrate the sensitivity of a prototype in vivo photoacoustic flowcytometry (PAFC) system described in Example 2 an experiment wasconducted using the prototype PAFC system to detect nanoparticlesintravenously injected into the tail veins of rats.

The in vivo measurements in this experiment were performed using the ratmesentery model. The rat (White Fisher, F344) was anesthetized usingketamine/xylazine at a dosage of 60/15 mg/kg, and the mesentery wasexposed and placed on a heated microscope stage, and bathed in Ringer'ssolution at a temperature of 37° C. and apH of 7.4. The mesenteryconsisted of transparent connective tissue of 7-15 μm thickness, and asingle layer of blood and lymph microvessels.

The nanoparticles used in this experiment were gold nanorods (GNR),obtained from the Laboratory of Nanoscale Biosensors at the Institute ofBiochemistry and Physiology of Plants and Microorganisms in Saratov,Russia. On the basis of TEM and dynamic light scattering analyses, theGNR were estimated to be approximately 15 nm in diameter andapproximately 45 nm in length on average. The nanoparticles were usedeither uncoated, or functionalized using thiol-modified polyethyleneglycol (PEG) (Liao and Hafner 2005).

A 250-μL suspension of GNR with a concentration of 10¹⁰ particles/ml wasinjected into the tail veins of three rats, followed by the continuousmonitoring of PA signals measured from 50-μm diameter blood vessels inthe rat mesentery using the PAFC system described in Example 2. PAFCmeasurements were taken using a laser fluence of 100 mJ/cm², a laserbeam diameter of approximately 50 μm, and a laser wavelength of 830 nm,near the maximum absorption of the GNR.

Uncoated GNR were rapidly cleared from the blood circulation within 1-3minutes preferentially by the reticuloendothelial system (data notshown). After PEGylated GNR injection, strong fluctuating PA signalsappeared with amplitudes significantly exceeding the PA backgroundsignals from blood vessels within the first minute and continued for14-25 minutes, depending on the individual animal. In addition, the PAbackground signal from the blood vessel increased approximately 1.5-2times above the pre-injection background levels, reaching a maximumlevel between four and nine minutes after injection, as shown in FIG. 5.

The averaged PA signals from three animals, measured for 15 minutesafter injection with GNR suspensions, are summarized in FIG. 6. Themaximum rate of individual PA signals per minute, representing thenumber of GNR in circulation, was achieved approximately 5 minutes afterinjection, with a gradual decrease in the signal rate over the next 10minutes.

The results of this experiment demonstrated that the prototype PAFCsystem possessed sufficient spatial and temporal resolution tocontinuously monitor the circulation of nanoparticles as small as 15 nmin diameter. In addition, the prototype PAFC system was sufficientlysensitive to track fluctuations of the concentration of circulatingparticles from the time that they were injected to the time that theparticles were cleared from circulation.

Example 4 Prototype In Vivo Photoacoustic Flow Cytometry System Used toDetect S. aureus Bacteria Circulating in Mice

To demonstrate the ability of the prototype photoacoustic flow cytometry(PAFC) system to detect bacteria cells in vivo under biologicalconditions, the following experiment was conducted. The prototype PAFCsystem, previously described in Example 2, was used to measure S. aureusbacteria in the circulation of nude mice.

The mouse ear model described in Example 2 was used for all measurementsof circulating bacteria in the experiment. Because the endogenous lightabsorption of S. aureus bacteria was relatively weak compared to theabsorption of other blood components in the NIR spectral range, thebacteria were labeled with the NIR-absorbing contrast substancesindocyanine green dye (ICG) and carbon nanotubes (CNT), due to theirrelatively high labeling efficiency and low toxicity (data not shown).

The S. aureus bacterium strain designated UAMS-1 was isolated from apatient with osteomyelitis at the McClellan Veterans Hospital in LittleRock, Ark., USA. The strain was deposited with the American Type CultureCollection and is available as strain ATCC 49230. UAMS-1 was cultured intryptic soy broth and grown aerobically for 16 h at 37° C. Cells wereharvested by centrifugation, resuspended in sterile PBS and incubatedwith Indocyanine Green (ICG) dye (Akorn Inc., USA) or carbon nanotubes(CNT) as described below.

Before incubation, ICG dye was filtered through a 0.22 μm pore sizefilter. A 150-μl aliquot of bacteria in suspension was incubated with375 μg of ICG in 150 μl of solution for 30 min at room temperature andthen for 2 h at 37° C. Labeled bacteria were centrifuged at 5,000 rpmfor 3 min and the resulting pellet was resuspended in PBS.

Single-walled and multi-walled carbon nanotubes (CNT) were purchasedfrom Carbon Nanotechnologies Inc. (Houston, Tex.) and Nano-lab Inc.(Newton, Mass.), respectively. The CNT samples used in this study wereprocessed using known methods (Kim et al. 2006). The average length anddiameter of the single-walled CNT were 186 nm and 1.7 nm respectively,and the average length and diameter of the multi-walled CNT were 376 nmand 19.0 nm respectively.

CNT solutions were treated with five cycles of 1.5 min of ultrasound ata power of 3 W followed by 0.5 min of rest, for a total of 10 minutes ofinterrupted ultrasound. A 150-μl aliquot of bacteria in suspension wasincubated with 150 μl of CNT solution for 30 minutes at room temperaturefollowed by 2 additional hours of incubation at room temperature.Labeled bacteria were centrifuged at 10,000 rpm for 5 min and theresulting pellet was resuspended in PBS.

Labeled 100-μl suspensions of S. aureus bacteria at a concentration of5×10⁵ cells/ml were injected into the mouse's tail vein, and theclearance of the labeled bacteria was monitored using PAFC measurementstaken from 50-μm diameter microvessels in the ears of mice. Laser energywas delivered at a wavelength of 805 nm for the S. aureus that waslabeled with ICG, and at a wavelength of 850 nm for the S. aureus thatwas labeled with CNT. For both label types, the laser energy wasdelivered at a beam diameter of approximately 50 μm and at a fluenceranging between 20 and 50 mJ/cm².

S. aureus bacteria labeled with ICG and CNT contrast substances yieldedsimilar results, summarized in FIG. 7. After injection of labeled S.aureus, PAFC detected a rapid appearance of bacteria in the ear bloodmicrovessels within the first minute, followed by a steady eliminationof the bacteria from the blood circulation over the next 3-5 minutes.Periodic PAFC monitoring of mice blood vessels over the next few daysrevealed that very rare bacteria labeled with CNT or possibly unattachedCNT continued to appear at an average rate of one PA signal every threeminutes, and the labeled bacteria or CNT was not completely cleared fromcirculation until about 60 hrs after the initial injection (data notshown).

The results of this experiment established the feasibility of PAFC forthe in vivo monitoring of individual cells in the circulatory systems ofliving organisms. Using appropriate contrast enhancement substances, thelaser fluence required for effective detection of cells in circulationwas well below the threshold levels for laser-induced cell damage.

Example 5 Prototype In Vivo Photoacoustic Flow Cytometry System Used toDetect E. coli Bacteria Circulating in Mice

To demonstrate the ability of the prototype photoacoustic flow cytometry(PAFC) system to detect bacteria cells in vivo under biologicalconditions, the following experiment was conducted. The prototype PAFCsystem, previously described in Example 2, was used to detect thebacteria E. coli strain K12, in the circulation of nude mice.

The mouse ear model described in Example 2 was used for all measurementsof circulating bacteria in the experiments described below. Because theendogenous light absorption of E. coli bacteria was relatively weakcompared to the absorption of other blood components in the NIR spectralrange, the bacteria were labeled with NIR-absorbing carbon nanotubes(CNT).

E. coli K12 strain was obtained from the American Type CultureCollection (Rockville, Md.) and maintained in Luria-Bertani (LB) medium,a solution consisting of 1% tryptone, 0.5% yeast extract, and 0.5% NaClat a pH of 7. A 100-μl aliquot of E. coli in PBS was incubated with 100μl of the CNT solution as described in Example 4 for 60 min at roomtemperature.

100-μl suspensions of CNT-labeled E. coli bacteria at a concentration of5×10⁵ cells/ml were injected into the mouse's tail vein, and theclearance of the labeled bacteria was monitored using PAFC measurementstaken from 50-μm diameter microvessels in the ears of mice. Laser energywas delivered at a wavelength of 850 nm, a beam diameter ofapproximately 50 μm and at a laser fluence of 100 mJ/cm². PAFCmeasurements, summarized in FIG. 8, detected a rapid appearance of thebacteria in circulation after injection, and the bacterialconcentrations in the blood decreased exponentially over the next 15-17minutes.

The results of this experiment confirmed the feasibility of PAFC for thein vivo monitoring of individual cells in the circulatory systems ofliving organisms. The laser fluence required for effective detection ofE. coli cells in circulation was well below threshold levels forlaser-induced cell damage.

Example 6 In Vivo PAFC Used to Detect Circulating Exogenous MelanomaCells

To demonstrate the ability to use in vivo PAFC to detect unlabeledmelanoma cells in circulation with extremely high sensitivity throughskin cells with varying levels of melanin pigmentation, the followingexperiment was conducted.

B16F10 cultured mouse melanoma cells (ATCC, Rockville, Md.) were used inthis experiment. The cells were maintained using standard procedures(Ara et al. 1990, Weight et al. 2006, Zharov et al. 2006), includingserial passage in phenol-free RPMI 1640 medium (Invitrogen, Carlsbad,Calif.) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Forcomparison to the detection of unlabelled melanoma cells, the endogenouscell absorption was increased by staining with ICG (Akorn Inc., USA), astrongly absorbent dye in the NIR range, for 30 min at 37° C. and in thepresence of 5% CO₂. No toxicity was observed after labeling as assessedusing the trypan blue exclusion assay (data not shown).

In vivo measurements of melanoma cells used the PAFC system previouslydescribed in Example 2 with a laser wavelength of 850 nm and a laserfluence of 80 mJ/cm². This wavelength falls within a region in which theabsorbance of melanoma cells is relatively high compared to theabsorbance of hemoglobin, a major component of blood, as determined byin vitro measurements summarized in FIG. 9.

To estimate the influence of endogenous skin melanin on PAFC detectionlimits, Harlan Sprague mice, strain: NIH-BG-NU-XID were used in thisexperiment. Female mice of this strain possess high levels skinpigmentation between 8 and 10 weeks of age. Mice were anaesthetized andplaced on the heated microscope stage as previously described in Example2.

A 200-μl volume of saline solution containing approximately 10⁵ mousemelanoma cells was injected into the mouse circulatory system through atail vein and then monitored using the PAFC system. The number ofmelanoma cells per minute detected using PAFC for melanoma cells afterinjection are summarized in FIG. 10 for melanoma cells with low melanincontent (FIG. 10A) and for melanoma cells with high melanin content(FIG. 10B). In the first 5 minutes of PA detection following intravenousinjection of cultured mouse melanoma cells, 600±120 PA signals(representing melanoma cells) per hour were observed, and the rate ofdetection of melanoma cells steadily decreased over the subsequent 20-30min. Approximately 20 cells/hour and 4 cells/hour were detected after 24h and 48 h of monitoring, respectively. The initial PA signal rate afterthe injection of melanoma cells stained with ICG contrast enhancementsubstances was 720±105 cells/hour (data not shown). Assuming that allstained melanoma cells were detected by in vivo PAFC, 82.4% of theunlabelled melanoma cells in circulation were detected by in vivo PAFCmeasurements.

The results of this experiment demonstrated the ability of in vivo PAFCto detect and monitor the appearance and progression of metastaticmelanoma cells in circulation non-invasively.

Example 7 In Vivo PAFC was Used to Detect Circulating SpontaneousMetastatic Cells During Tumor Progression

An experiment was conducted to determine the ability of in vivo PAFC todetect relatively scarce endogenous metastatic melanoma cellscirculating in lymph vessels. The PAFC system described in Example 2 wasused to monitor endogenous metastatic melanoma cells in mice. The lasercharacteristics used in this experiment are identical to those describedin Example 5.

Nude mice were anaesthetized and placed on the heated microscope stageas previously described in Example 2. The ear blood vessels underexamination were located 50-100 μm deep and had diameters of 35-50 μmwith blood velocities of 3-7 mm/sec. To increase the probability ofdetection of rare metastatic cells, blood vessels with relatively largediameters of 150-300 μm and flow velocities up to 10-30 mm/s in the skinof the abdominal wall were examined using a customized skin foldchamber.

50-μl suspensions containing 10⁶ B16F10 cultured mouse melanoma cells(ATCC, Rockville, Md.) were subcutaneously injected into nude mice.Melanoma tumors subsequently formed in the ears of the mice and in theskin on the backs of the mice. PAFC was performed on ear and abdominalblood vessels to monitor the circulatory system for the appearance ofmetastatic cells, and PA mapping, described below, was used to monitorthe growth of tumors.

During ear tumor development, individual or groups of melanoma cellswere first detected in the skin area close to the tumor site on thesixth day following tumor inoculation using PA mapping measurements. PAmapping measurements utilized PA signals derived by scanning a focusedlaser beam with diameter of 10 μm across ear. Metastatic cells firstappeared in ear microvessels near the tumor on the twentieth day afterinoculation at a rate of 12±5 cells/hour (data not shown). Surprisingly,during the same time period, no melanoma cells had yet been detected inthe abdominal skin blood vessels. 25 days after inoculation, the averagecount of melanoma cells detected in the ear veins increased to 55±15cells/hour, and at this same time, melanoma cells were detected inabdominal wall skin vessels at a rate of 120±32 cells/hour. Thirty daysafter inoculation, the detection rate decreased to 30±10 cells/hour inthe abdominal vessel, which may be attributed to inhibition ofmetastatic cell production in the primary tumor. PA mapping of selectedtissue and organs revealed multiple micrometastases in cervical andmesenteric lymph nodes, as well as in lung and liver tissues.

PAFC measurements of the nude mouse back tumor model revealed theappearance of metastatic melanoma in abdominal skin blood vessels closeto the tumor site on day 5, much earlier than in the tumor ear model.This indicates a much greater likelihood of detecting the initialmetastatic process in the vicinity of the primary tumor.

Thirty days after tumor inoculation, the average concentration ofmelanoma cells was 150±39 cells/ml, corresponding to a circulating rateof approximately 4-10 cells/min in a 50-mm blood vessel and a flowvelocity of 5 mm/s. The ultimate PAFC threshold sensitivity of the nudemouse back tumor model was estimated as 1 cell/ml. This circulating ratecorresponded to an incidence of approximately one melanoma cell among100 million normal blood cells.

The results of this experiment indicated that in vivo PAFC and PAmapping were sensitive methods with which to monitor the development ofmetastasized melanoma cells non-invasively, with high sensitivity andaccuracy.

Example 8 In Vivo PAFC was Used to Detect Spontaneous Metastatic Cellsin Lymphatic Vessels During Tumor Progression

To determine the feasibility of detecting individual metastatic cells inlymph flow, the following experiment was conducted. A photoacoustic flowcytometer (PAFC) was used to monitor lymph flow for the presence of WBC,RBC, and metastatic melanoma cells.

The animal models used in this experiment were nu/nu nude mice, weighing20-25 g (Harlan Sprague-Dawley). PAFC measurements were taken using thelymphatic vessels in the ears using a heated platform as described inExample 2. Melanoma tumors in the ear and back skin of the mice wereinduced by the subcutaneous injection of B16F10 mouse melanoma cells asdescribed in Example 6.

To locate the lymphatic vessels in the mouse ear, a PA mapping processusing a PA contrast agent was used. Ethylene blue (EB) dye, commonlyused for lymphatic research, was injected into the lymphatic vesselwalls. A 639 nm laser beam was then used to illuminate the lymphaticvessel at a wavelength of 639 nm, corresponding to the maximumabsorption of EB dye, and the resulting PA signal emitted by the EB dyewas monitored. The position of the laser beam on a lymph vessel wasfixed when the PA signal amplitude reached its maximum at the laserwavelength of 639 nm.

In vivo PAFC detection of unlabeled melanoma cells relied on melanin asan intrinsic cell marker, as in Example 7. Melanoma cells were detectedusing a laser wavelength of 850 nm, a laser fluence of 35 mJ/cm², and alaser beam diameter of approximately 50 μm. In mice with induced skinmelanomas, metastatic cells were observed to appear in a lymphaticvessel of the mouse's ear on the fifth day after inoculation at a rateof 1.2±0.5 cells/min, which steadily increased over the course of 2weeks (data not shown). In mice with a melanoma tumor in the ear,melanoma cells appeared in skin lymphatics 20 days after inoculation. 30days after inoculation strong PA signals detected the presence ofmetastatic melanoma cells in the sentinel lymph nodes, which was laterconfirmed by histology (data not shown). FIG. 11 shows the PA signalsdetected from single metastatic melanocytes circulating in the lymphaticvessel in the mouse ear five days after tumor inoculation.

The results of this experiment demonstrated the feasibility of detectingrelatively scarce metastatic melanoma cells circulating in the lymphaticsystem using in vivo PAFC techniques, with high sensitivity andaccuracy.

Example 9 In Vivo PAFC was Used to Detect Red Blood Cells andLymphocytes Simultaneously Circulating in Lymph Vessels

To determine the feasibility of detecting unlabeled individual cells ofdifferent types circulating in lymph flow, the following experiment wasconducted. A photoacoustic flow cytometer (PAFC) was used to monitorlymph flow for the presence of red blood cells and lymphocytes.

The animal models used in this experiment were 150-200 g rats (HarlanSprague-Dawley). PAFC measurements were taken using lymphatic vessels inthe mesentery of the rat, using the method described in Example 3.Lymphatic vessels were located, and the laser was focused on thelymphatic vessel using the methods described in Example 8.

Spectroscopic studies in vitro revealed that PA signals from lymphocytesreached maximal amplitude in the visible-spectral range near 550 nm,associated with cytochrome c acting as an intrinsic absorption marker(data not shown). Background PA signals from vessels and surroundingtissues were approximately 4-6-fold less than from single lymphocytes atthis wavelength due to the low level of background absorption and laserfocusing effects.

The in vitro PAFC system described in Example 2 was used to detectcirculating cells in the lymphatic vessels of the rat mesentery. Thelaser used in the PAFC system had a wavelength of 550 nm and a fluenceof 100 mJ/cm², and a circular beam diameter of approximately 50 μm. Thecell detection rate obtained in lymphatic vessels was 60±12 cells/min. Agraph showing the PA signals detected by the PAFC system in a ratmesentery lymphatic vessel is shown in FIG. 12. Lymphocyte heterogeneityresulted in 2-2.5-fold fluctuations in PA signal amplitude from cell tocell. A small fraction of the detected cells had strong PA signalamplitudes exceeding those of the lymphocyte signals by a factor of 10to 20-fold. One such strong PA signal is shown as a white bar in FIG. 12at 28 seconds. Subsequent spectral and imaging analysis identified raresingle red blood cells (RBCs) as the sources of these excessively strongPA signals.

The results of this experiment demonstrated that the in vivo PAFC systempossessed sufficient sensitivity and accuracy for the simultaneousdetection of red blood cells and lymphocytes circulating in thelymphatic vesicles.

Example 10 In Vivo Two-Wavelength PAFC was Used to Discriminate Between3 Different Exogenously Labeled Cell Types in Circulation within LymphVessels

To demonstrate the ability of the photoacoustic flow cytometry (PAFC)system to detect cells using more than one wavelength of laser light,the following experiment was conducted. In this experiment, a PAFCsystem was used to detect exogenous blood cells that were labeled withthree different nanoparticles, while circulating in blood vessels (datanot shown) and in lymphatic vessels. The PAFC system detected the cellsby illuminating the cells with laser pulses of two different wavelengthsin the near-infrared (NIR) spectrum.

A PAFC system similar to that described in Example 2 was used to detectthe circulating cells. However, in the PAFC system used in thisexperiment, the laser of the PAFC system pulsed light at two differentwavelengths, corresponding to the wavelengths of maximum absorption fortwo of the nanoparticles used to label the cells. The first laser pulsewas at a wavelength of 865 nm, a laser fluence of 35 mJ/cm², and pulseduration of 8 ns. 10 μs after the end of the first laser pulse, a secondlaser pulse was delivered at a wavelength of 639 nm, a laser fluence of25 mJ/cm², and pulse duration of 12 ns. The paired laser pulses wererepeated at a frequency of 10 Hz.

The animal models used in this experiment were nu/nu nude mice, weighing20-25 g (Harlan Sprague-Dawley). PAFC measurements were taken usinglymphatic vessels in the mesentery of the mouse, using the methodsdescribed in Example 3. Lymphatic vessels were located, and the laserwas focused on the lymphatic vessel using the methods described inExample 8.

Normal fresh blood cells were obtained from heparinized whole-bloodsamples of donor mice after terminal blood collection. Red blood cellswere isolated by simple centrifugation, and lymphocytes were isolated byHistopaque (Sigma-Aldrich) density gradient centrifugation asrecommended by the supplier.

The nanoparticles used to label the various blood cells used in thisexperiment were gold nanorods (GNR) and gold nanoshells (GNS), providedby The Laboratory of Nanoscale Biosensors at the Institute ofBiochemistry and Physiology of Plants and Microorganisms in Saratov,Russia. The GNR had an average diameter of 16 nm, an average length of40 nm, and a relatively narrow absorption band of 660±50 nm. The GNS hadan average diameter of 100 nm, and a maximum absorption near 860 nm.Both GNR and GNS were coated with polyethylene glycol in the processdescribed in Example 3. Single-walled CNT with an average length of 186nm and an average diameter of 1.7 nm were purchased from CarbonNanotechnologies Inc. CNT absorb laser energy over a wide range ofwavelengths with an efficiency that monotonically decreases aswavelength increases. All particles were in suspension at aconcentration of about 10¹⁰ nanoparticles/ml.

Live neutrophils were labeled with the GNS, live necrotic lymphocyteswere labeled with the GNR and apoptotic lymphocytes were labeled withthe CNT. The cells were labeled by incubating 100-μl aliquots of eachcell type in phosphate-buffered saline with 100 μl of CNT, GNR, or GNSfor 15 min at room temperature.

The labeled cells, mixed in approximately equal proportions, wereintravenously injected into the tail vein of the mouse. After 6 h,mesenteric lymphatics were illuminated with two laser pulses atwavelengths of 865 nm and 639 nm as described above. PA signals at arate of 1-3 signals/min were detected at this time.

The PA signals had one of three distinctive temporal shapes associatedwith the response of the three different labels to the paired laserpulses, shown in FIG. 13. PA signals from necrotic lymphocytes markedwith GNR were generated in response to the 639 nm laser pulse only,after a 10-μs delay, as shown in FIG. 13A. The apoptotic lymphocytesmarked with GNS generated PA signals in response to laser pulse at awavelength of 865 nm with no delay, as shown in FIG. 13B. Liveneutrophils marked with CNT generated two PA signals after a 10-μsdelay, as shown in FIG. 13C. One signal was generated in response to the639 nm laser pulse, and the second PA signal was generated in responseto the 850-nm laser pulse, due to comparable CNT absorption at bothwavelengths.

The results of this experiment demonstrated that with the use of variouscontrast substances and two wavelength cell identification techniques,the in vivo PAFC apparatus detected and discriminated between liveneutrophils, necrotic lymphocytes, and apoptotic lymphocytes that werecirculating in the lymphatic vessels. This method may also be extendedto unlabelled cells circulating in the lymphatic or circulatory systems,using a pair of laser pulse wavelengths selected to generate a unique PAsignal shape for each cell type to be detected.

Example 11 Spatial Resolution and Maximum Detectable Vessel Depth of aPrototype In Vivo PAFC System was Assessed

To determine the maximum spatial resolution and maximum detectablevessel depth of the PAFC system, the following experiment was conductedusing the prototype PAFC system described in Experiment 2 and the mouseear model with circulating melanoma cells, as described in Example 7.Mouse melanoma cells were injected into the tail veins of nude mice andPAFC measurements were conducted as described in Example 7.

The PAFC system achieved a lateral resolution of 5-15 μm when detectingmelanoma cells circulating in mouse ear blood vessels with diameters of10-70 μm at depths of 50-150 μm. However, when melanoma cellscirculating in mouse ear blood vessels at a depth of 0.5 mm weremeasured, the lateral resolution decreased to 30-50 μm due to thescattering of the 850 nm laser pulses by the additional tissue betweenthe PAFC laser and the targeted blood vessels.

The maximum potential of the PAFC to detect cells circulating in deepvessels was estimated by overlaying layers of mouse skin of varyingthickness over intact mouse skin containing peripheral blood vessels ata depth of approximately 0.3 mm below the surface of the intact skin.Using the PAFC system described in Example 2 with an unfocusedultrasound transducer (Panametrics model XMS-310, 10-MHz), PA signalswere detected at total skin thicknesses up to approximately 4 mm, with a27-fold signal attenuation due to light scattering. When a focusedultrasound transducer was used (Panametrics model V316-SM, 20 MHz, focallength 12.5 mm), PA signals were detected from melanoma cellscirculating in the mouse aorta at a depth of approximately 2.5 mm,resulting from a laser pulse wavelength of 850 nm. Even at a totaltissue depth as high as 11 mm, the PA signals emitted by circulatingmetastatic melanoma cells illuminated by 532 nm laser pulses remaineddiscernible from the background PA signals from surrounding tissues. Thelateral resolution at this vessel depth, measured by changing the angleof the ultrasonic transducer, was estimated to be approximately 250 μm(data not shown).

The results of this experiment demonstrated that the PAFC system wascapable of detecting circulating melanoma cells at a vessel depth of upto 11 mm with a resolution of approximately 250 μm. This resolution maybe improved significantly through the use of higher frequency ultrasoundtransducers, such as 50 MHz transducers.

Example 12 The Sensitivity of the Spatial Resolution of a Prototype InVivo PAFC Device to Skin Pigmentation Levels was Assessed Using the NudeMouse Model

To determine the sensitivity of the PAFC system to the level of skinpigmentation, the following experiment was conducted. The PAFC devicedescribed in Example 2 was used to measure PA signals from blood vesselsin nude mice skin with low and high levels of pigmentation using methodssimilar to those described in Example 7.

In the low-pigmented nude mouse model, the background PA signal fromskin cells was very weak. PA signals measured by a high frequencyultrasound transducer (Panametrics model V-316-SM, 20 MHz) resultingfrom the simultaneous irradiation of two circulatory vessels at depthsof approximately of 0.3 mm and 2.4 mm, were determined to have a timeseparation of approximately 1.4 ms. This delay is consistent withsignals emitted by cells with a separation distance of 2.1 mm, assuminga velocity of sound in soft tissue of approximately 1.5 mm/ms. Similarresults were obtained for measurements of circulatory vessels in thehighly pigmented nude mouse model (data not shown).

The results of this experiment demonstrated that the level of skinpigmentation did not significantly affect the spatial resolution of thePAFC device. For strongly pigmented skin, the assessment of deepervessels may actually be enhanced because the skin pigmentation mayfacilitate the discrimination between PA signals from circulatingindividual cells and PA signals from the skin.

Example 13 Methods of Enriching the Incidence of Circulating MetastaticCells Measured by PAFC In Vivo were Demonstrated Using the Mouse EarModel

To determine the feasibility of novel methods for increasing theconcentrations of circulating metastatic cells detected by the in vivoPAFC device, the following experiment was conducted. Using the mouse earmodel to measure the incidence of circulating metastatic melanoma cells,as described in Example 7, the effect of gentle mechanical squeezing ofblood microvessels was assessed. This method of enriching the localincidence of rare circulating cancer cells in vivo exploited the sizedifferences between melanoma cells (16-20 mm), WBC (7-8 mm), and RBC(5-6 mm) and the high deformability of RBC compared to cancer cells. Thelumen size of the microvessel was decreased to 10-15 μm through gentlemechanical squeezing of blood microvessels in 50-μm microvessels ofmouse ear. After squeezing a 50-μm mouse ear blood vessel for 10 min,then quickly releasing the vessel, the rate of metastatic melanoma cellsmeasured by PAFC immediately after vessel release increasedapproximately 8-fold, relative to the rate measured before squeezing.The degree of blood vessel squeezing could be controlled by monitoringincreases and decreases in PA signal amplitudes.

The results of this experiment demonstrated that local enrichment ofcirculating metastatic melanoma cells was achieved through themechanical restriction of circulatory vessels.

Example 14 The Background Absorption by Surrounding Blood Cells wasManipulated by Changes in Blood Oxygenation, Hematocrit, and BloodOsmolarity

To determine the effects of changes in blood oxygenation, hematocrit,and osmolarity on the background absorption of blood cells during invivo PAFC, the following experiment was conducted. The absorption oflaser energy by hemoglobin in its oxygenated (HbO₂) and deoxygenated(Hb) forms differs, depending on the oxygen saturation state of thehemoglobin and the wavelength of the laser pulse. The total absorptionof red blood cells decreases as oxygenation increases for laser pulsewavelengths 810-900 nm, and the absorption of red blood cells decreaseswith increasing blood oxygenation at laser pulse wavelengths of 650-780nm. Thus, the oxygenation of the red blood cells can be manipulated tominimize the background PA signals emitted by the red blood cells.

Pure oxygen was delivered to a mouse using a mask around the mouse'shead, and the background PA signal obtained before and after theincreased blood oxygenation was measured using the in vivo PAFC systemdescribed in Example 2. The increased blood oxygenation resulting fromthe exposure of the mouse to pure oxygen for 15 minutes caused thebackground PA signal from veins to decrease by a factor of 1.36±0.14,using a laser pulse wavelength of 750 nm. Replacing the delivery of pureoxygen with the delivery of pure nitrogen led to a 35% decrease inbackground PA signal in an arteriole at a laser pulse rate of 900 nm.

Another experiment was conducted to assess the effects of decreasing thedensity of the circulating RBC as measured by the hemotocrit on thebackground signal from circulating red blood cells. The hemotocrit of amouse's blood was temporarily reduced by the intravenous injection of0.5 ml of standard saline solution into the vein tail. After the salineinjection, PA signals from a 50-μm ear mouse vein dropped by a factor of2.3±0.3, and nearly returned to initial levels within about 1.5 minutes.

Blood osmolarity causes an increase in the RBC volume (swelling) thatresulted in a decrease in the average intracellular Hb concentration.Injection of 100-mL of hypertonic NaCl solution into the mouse tail veinled to an approximately 2-fold decrease in the PA signal in the earvein.

The results of this experiment demonstrated that the background PAsignals resulting from the emission of PA signals by red blood cells maybe minimized by manipulation of the chemical environment of the blood,including blood oxygenation, hemotocrit, and blood osmolarity. Theseapproaches may be readily applicable to human subjects because theprocedures used in this experiment are routinely used in clinicalpractice.

Example 15 Microbubbles Conjugated with Nanoparticles were Assessed as aContrast Agent for PAFC

To assess the effectiveness of microbubbles conjugated withnanoparticles as a contrast agent in PAFC, the following experiment wasconducted. Microbubbles (Definity Inc.) with average diameters of 2-4 μmwere incubated with PEG-coated gold nanoshells (GNS), previouslydescribed in Example 10 for 1 hr at room temperature. The measurement ofPA signals in vitro, as described in Example 1 was conducted formicrobubbles only, GNS only and microbubbles conjugated with GNS. Themicrobubbles conjugated with GNS emitted the strongest PA signals, theGNS only emitted somewhat weaker PA signals, and the microbubbles aloneemitted negligible PA signals (data not shown).

Increasing the energy of the laser pulses illuminating theGNS-conjugated microspheres led to a dramatic increase of the emitted PAsignals, followed by the disappearance of the microbubbles after asingle laser pulse. This observation was attributed to the laser-inducedoverheating of the GNS leading to a dramatic temperature increase of thegas trapped inside of the microbubbles that ultimately ruptured themicrobubbles.

The results of this experiment demonstrated that microbubbles conjugatedwith GNS were an effective contrast agent, but that the energy of thelaser pulses must be carefully moderated to avoid bursting themicrobubbles. Because the microbubbles may be selectively attached toblood clots or taken up by activated white blood cells, this contrastagent may expand the potential applications of in vivo PAFC to includethe detection of blood clots and certain activated white blood cells.

Example 16 Two-Wavelength In Vivo PAFC Used to Detect CirculatingExogenous Melanoma Cells

To demonstrate the ability to use two-wavelength in vivo PAFC to detectinjected unlabeled melanoma cells in circulation with extremely highsensitivity, the following experiment was conducted. B16F10 culturedmouse melanoma cells (ATCC, Rockville, Md.) were obtained and maintainedas described in Example 6. The experiments were performed using a nudemouse ear model similar, described in Example 2 (n=25). To mimicmetastatic cells, approximately 10⁵ tumor-derived B16F10 cells in a100-μl volume of saline solution were injected into the mousecirculatory system through a tail vein and then monitored in an ear veinusing an apparatus and methods similar to those described in Example 10.An ear blood vessel was illuminated by two laser pulses at wavelengthsof 865 nm and 639 nm with a 10-ms delay between the pulses.

The melanoma cells were distinguished from surrounding blood cells,based upon the distinctive absorption spectra of the melanoma cells, asdescribed previously in Example 6 and summarized in FIG. 9. Melanomacells emitted two PA signals with a 10-ms delay, corresponding to thetwo laser pulses. The first PA signal, induced by the 639 nm laserpulse, had a higher amplitude than the PA signal induced by the 865 nmpulse, as shown in FIG. 14A. Red blood cells, the most numerous bloodcells in circulation, generated two PA signals with lower amplitudesthan the corresponding PA signals generated by the melanoma cells. Inaddition, for the red blood cells, the amplitude of the PA signalinduced by the 865 nm pulse was slightly higher than the PA signalinduced by the 639 nm laser pulse, as shown in FIG. 14B.

The PA signals corresponding to the melanin particles were cleared overa two-hour period following the injection, as shown in FIG. 15.

Based on comparisons to similar data measured for melanoma cells labeledwith markers that emitted strong PA signals, it was estimated thatapproximately 89% of the unlabelled melanoma cells were detected (datanot shown). This percentage was lower than that found in previous invitro studies (96%) and indicated a false-negative-signal rate of 1.5cells/min because of the influence of background absorption by RBCs(data not shown). Longer-term monitoring of PA signals from ear bloodvessels without prior melanoma cell injection detected no false-positivesignals using as its criteria a signal-to-noise ratio ≥2, where thesignal noise was associated with fluctuations of laser energy and thedensity of red blood cells in the detected volume.

The results of this experiment demonstrated that two-color in vivo flowcytometry was an effective method of detecting metastatic melanoma cellsin circulation. It was estimated that the method described abovedetected approximately 89% of the melanoma cells in circulation, withslightly lower detection rates due to skin pigmentation.

Example 17 Two-Wavelength In Vivo PAFC was Used to Detect CirculatingSpontaneous Metastatic Cells During Tumor Progression

An experiment was conducted to determine the ability of two-wavelengthin vivo PAFC to detect relatively scarce endogenous metastatic melanomacells circulating in lymph vessels. The PAFC system described in Example10 was used to monitor endogenous metastatic melanoma cells in mice.Tumors were induced in nude mice by subcutaneous injections of melanomacells using methods similar to those described in Example 7. Tumorsformed and proliferated in the skin of the ear and the back of the nudemice over a period of 4 weeks, as previously described in Example 7.

PAFC was used to count spontaneous metastatic melanoma cells in an ˜50μm-diameter ear blood vessel and a 100-200-μm-diameter skin blood vesselduring tumor progression in the ear and skin of a mouse, as summarizedin FIG. 16. As previously described in Example 7, the skin tumor growthrate was faster than that of the ear tumors, and metastatic melanomacells appeared more quickly in the circulation, as indicated by the meancell detection rate measured in the skin capillaries, shown as solidsquare symbols in FIG. 16. In particular, within the first week afterthe induction of the tumors, about 1-4 melanoma cells/min were detectedin the skin vasculature, and as the tumor size increased, the rate ofdetection of metastatic melanoma cells gradually increased to about 7cells/min and about 12 cells/min after 3 weeks and 4 weeks,respectively.

The results of this experiment indicated that in vivo PAFC and PAmapping were sensitive methods with which to monitor the development ofmetastasized melanoma cells non-invasively, with high sensitivity andaccuracy.

Example 18 PAFC System was Used to Determine Photoacoustic Response ofQuantum Dot Markers In Vitro

An experiment was conducted to determine the ability of two-wavelengthin vivo PAFC to detect quantum dot cell markers in vitro. The PAFCsystem described in Example 2 was used to measure photoacoustic pulsesemitted by quantum dots in response to laser pulses with wavelengths of625 nm, pulse widths of 8 ns, and laser fluences ranging 0.001-10 J/m².The laser beam used to pulse the quantum dots had a diameter of about20-30 μm in the sample plane. Quantum dots were obtained commerciallywith a polymer coating as well as with a streptavidin protein coating(Qdot 655 nanocrystals, Invitrogen, Carlsbad Calif.). The quantum dotshad diameters of about 15-20 nm and an emission wavelength of about 655nm. Either single or aggregations of quantum dots were diluted with abuffer of 2% BSA/PBS and mounted in a layer of less than 1 μm on amicroscope slide.

The PAFC system was used to pulse the quantum dot preparation with laserfluences ranging from 0.001-30 J/m². The magnitudes of the PA signalsemitted by the quantum dots are summarized in FIG. 17. The PA signalresponse of the quantum dot preparations had a non-linear response tothe variations in laser fluences. PA signal amplitude graduallyincreased in the laser fluence range from 0.1-1 J/cm². Through the laserfluence range between 1.5-7 J/cm², the response increased dramaticallyin a non-linear manner, and continued to increase in magnitude up to alaser fluence of 15 J/cm². At laser fluences above 15 J/m², theresponses of the quantum dot preparations were saturated.

The PA signal response as a function of the number of laser pulses forlaser fluences of 1.2, 4.0, 6.2, and 12.4 J/cm² are summarized in FIG.18. There was no sign of alteration of the laser pulse-induced PAsignals at laser fluences below 3 J/cm², indicating no blinkingbehavior, unlike the stereotypical fluorescent blinking behaviorobserved in quantum dots. At higher laser fluences, significantdecreases in the PA signal amplitude were observed with an increase inthe number of pulses, possibly due to laser induced melting ofthermal-based destruction by explosion of the quantum dots.

The results of this experiment indicated the quantum dots generatedstrong PA signals in response to laser pulses.

Example 19 PAFC System was Used to Detect Bacteria and Melanoma CellsMarked with Magnetic Nanoparticles In Vivo

To demonstrate the application of magnetic nanoparticles asphotoacoustic (PA) contrast agents, the following experiment wasconducted.

S. aureus bacteria, described in Example 4, and melanoma cells,described in Example 6, were labeled with super paramagnetic iron oxidenanoparticles (Clementer Associates, Madison, Conn.). The nanoparticlesconsisted of a 50-nm core of magnetite (Fe₃O₄), coated with a 10-15 nmlayer of Dextran and fluorescent dye. Both bacterial cells and melanomacells were cultured at a density of approximately 10⁶ cells/mL, andmagnetic nanoparticles were added to the cell cultures at a density of0.5 mg/mL, and loaded into the cells by endocytosis for a minimum of 1hour at 37° C. Labeled cells were centrifuged at 5,000 rpm for 3 minutesand the resulting pellet were resuspended in PBS.

The photoacoustic flow cytometry system (PAFC) system was similar indesign to the PAFC system previously described in Example 2, withmodifications to the laser, amplifier, and transducer components. Adiode laser 905-FD1S3J08S (Frankfurt Laser Company) with driver (IL30C,Power Technology Inc, Little Rock, Ark.) was used to pulse the unboundmagnetic nanoparticles and labeled cells with a pulse width of 15 ns,and a pulse repetition rate of 10 kHz. The laser beam dimensions used topulse the nanoparticles and cells had an elliptical cross-section withminor and major axis dimensions of 11 μm and 75 μm, respectively, and afluence energy maximum of 0.6 J/cm². The laser-induced PA signals weredetected by a 5.5 mm-diameter, 3.5 MHz ultrasound transducer (model6528101, (masonic Inc., Besancon, France), amplified using a 2 MHzamplifier (Panametrics model 5660B) and recorded with a Boxcar datarecorder (Stanford Research Systems, Inc.) and a Tektronix TDS 3032Boscilloscope.

To determine the clearance rate of unbound magnetic nanoparticles, thenude mouse ear model described in Example 2 was used. A 100-mL PBSsuspension of the magnetic nanoparticles at a concentration of about10¹¹ nanoparticles/mL was injected into the vein tail of the mice.

The magnetic nanoparticles were detected using the PAFC system describedabove. The laser pulses were delivered to the unbound magneticnanoparticles at a wavelength of 639 nm and a laser fluence of 1.5J/cm². The detection and subsequent clearance of the magnetic particlesin the nude mouse ear model are summarized in FIG. 19. PA signalscorresponding to the magnetic nanoparticles appeared within the firstminute after injection. The PA signals were a combination of afluctuating continuous PA background with superimposed large-amplitudePA signals. The magnitude of the background signal associated with themagnetic nanoparticles exceeded the PA background signals from the bloodvessels by a factor of 2-3. The stronger but less frequentlarge-amplitude PA signals may be associated with random fluctuations ofthe number of magnetic nanoparticles in the detected volume andappearance of small aggregates of magnetic nanoparticles. The clearancetime of the magnetic nanoparticles from the mouse ear microcirculationwas in the range of 10-20 minutes.

Approximately 10⁵ B16F10 melanoma cells or S. aureus labeled withmagnetic nanoparticles in 100 μL of saline solution were injected into amouse tail vein and then monitored in the mouse ear using the PAFCsystem described above. Labeled melanoma cells were detected using a 905nm, 0.4 J/cm² laser pulse, and the bacterial cells were detected usingan 850 nm, 0.9 J/cm² laser pulse. The resulting PA signals emitted by S.aureus and melanoma cells labeled with magnetic nanoparticles aresummarized in FIG. 20. Numerous PA signals from individual circulatingcells were detected, with a maximum rate of detection within the first1-3 minutes. The average half-life of the labeled bacteria and cancercells in the blood microcirculation was 4.5 and 12 min, respectively.

After the labeled melanoma cells and bacteria were essentially clearedfrom the circulation and only rare PA signals were detected, a localpermanent magnetic field was imposed through intermediate tissue to theblood microvessels. The local permanent magnetic field was provided by acylindrical Neodymium-Iron-Boron (NdFeB) magnet with Ni—Cu—Ni coatingthat was 3.2 mm in diameter and 9.5 mm long with a surface fieldstrength of 0.39 Tesla (MAGCRAFT, Vienna, Va.). The distance between themagnet and the microvessel walls ranged between 50-100 μm. As shown inFIGS. 21 and 22, the application of the magnetic field to the bloodmicrovessels led to an immediate increase in both PA signal amplitudesand rate of detection in the vicinity of the magnet for the labeledmelanoma cells and bacterial cells respectively.

The results of this experiment demonstrated that magnetic nanoparticlescould be used to label circulating melanoma and bacteria cells for usein the prototype PAFC system. Further, a magnetic field applied to theblood microvessel in which the PAFC detected circulating cells was ableto locally enrich the concentration of cells to be detected.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made thereto without departing from the spirit and scope of theinvention as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this inventionas defined in the claims appended hereto.

REFERENCES

-   1. Ara, G. Anderson, R. R., Mandel K. G., Ottesen, M, Oseroff, A.    R., (1990), “Irradiation of pigmented melanoma cells with high    intensity pulsed radiation generates acoustic waves and kills    cells.” Lasers Surg Med, 10, 52-59.-   2. Kim, J. W., Kotagiri, N., Kim, J. H., and Deaton, R, (2006), “In    situ fluorescence microscopy visualization and characterization of    nanometer-scale carbon nanotubules labeled with 1-pyrenebutanoic    acid, succinimdylester,” Appl. Phys. Lett., 88, 213110.-   3. D. O. Lapotko and V. P. Zharov (2005). “Spectral evaluation of    laser-induced cell damage with photothermal microscopy,” Laser Surg.    Med. 36, 22-33.-   4. H. Liao and J. H. Hafner (2005). “Gold nanorod bioconjugates,”    Chem. Mater. 17, 4636-4641.-   5. Weight, R. M., Viator, J. A., Dale, P. S., Caldwell, C. W.,    Lisle, A. E. (2006), “Photoacoustic detection of metastatic melanoma    cells in the human circulatory system.”, Opt Lett. 31, 2998-3000.-   6. V. P. Zharov and D. O. Lapotko (2005). “Photothermal imaging of    nanoparticles and cells (review),” IEEE J. Sel. Topics Quant.    Electron. 11, 733-751-   7. Zharov, V. P., Galanzha, E. I., Tuchin, V. V. (2006), “In vivo    photothermal flow cytometry: imaging and detection of individual    cells in blood and lymph flow.”, J Cell Biochem. 97, 916-932.

What is claimed is:
 1. A method of detecting individual moving targetobjects in fast flowing blood or lymphatic vessels of a living organismat up to 15 cm away from the surface of the skin, the method comprising:a. pulsing an individual fast moving target object with a repeatingseries of at least two laser pulses, wherein the at least two laserpulses comprise a first laser pulse with a first wavelength and a secondlaser pulse with a second wavelength different from the first wavelengthand the second laser pulse is separated from the first laser pulse by atime delay ranging between about 0.1 μs and about 100 μs, wherein thefirst wavelength is between about 220 and about 2500 nm and the secondwavelength is at a different wavelength between about 220 and about 2500nm, wherein the first wavelength and the second wavelength are eachemitted at a pulse width ranging between about 0.1 ps and about 1000 ns,a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, anda pulse energy fluence ranging between about 0.1 mJ/cm² and about 1000J/cm²; b. detecting at least two photoacoustic pulses emitted by theindividual fast moving target object in response to the at least twolaser pulses at a sample rate ranging between about 10 kHz and about 100MHz; and, c. analyzing the at least two photoacoustic pulses todetermine at least one characteristic of the target object selected froma type of target object, quantity of target objects, a concentration oftarget objects, a flow speed of the target objects, a total bloodvolume, and combinations thereof, wherein the time delay between thefirst laser pulse and the second laser pulse ensures that (i) the atleast two laser pulses illuminate the same individual fast moving targetobject and (ii) the second laser pulse does not reach the individualfast moving target object prior to detection of the photoacoustic pulseinduced by the first laser pulse.